Methods for treating metabolic diseases by inhibiting myostatin activation

ABSTRACT

The present invention relates to antibodies, or antigen-binding fragments thereof, that specifically bind proMyostatin and/or latent Myostatin, and methods and uses thereof for treating metabolic diseases.

RELATED APPLICATIONS

This application claims priority to the following patent applications:U.S. provisional application 62/443,455 and EP priority application17150586.0, each filed on Jan. 6, 2017, U.S. provisional application62/530,311 filed on Jul. 10, 2017, and U.S. provisional application62/608,069, filed on Dec. 20, 2017. The entire contents of these priorapplications are incorporated herein by reference, including theSequence Listing, which was submitted electronically in ASCII format.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 5, 2018, isnamed “SR16-WO-PCT_Sequence_Listing_127036-00220.txt” and is 108,914bytes in size.

BACKGROUND OF THE INVENTION

Metabolic diseases affect millions of people worldwide, and patientswith metabolic diseases generally experience a loss of fat-free or leanmuscle mass, an excess gain of fat mass, a lower metabolic rate, insulinresistance, lack of ability to regulate blood sugar, weight gain, andincrease in body mass index. Thus, these patients are at risk ofdeveloping major complications, such as diabetes, obesity, coronaryartery disease, hypertension, stroke, atherosclerosis, heart failuresuch as chronic heart failure (CHF), including congestive heart failure,metabolic bone disorders, gallbladder disease, osteoarthritis, sleepapnea, reproductive disorders such as polycystic ovarian syndrome,cancers of the breast, prostate, and colon, and increased incidence ofcomplications of general anesthesia.

In addition to the serious health consequences of these metabolicdiseases, serious economic costs are associated with these diseases. Forexample, the total cost of treating diabetes and its complications inthe United States has been estimated at $245 billion annually. Theestimated annual health care costs of obesity-related illness are astaggering $190.2 billion or nearly 21% of annual medical spending.Substantial costs to both society and its citizens are incurred not onlyfor direct costs of medical care for these metabolic diseases, but alsofor indirect costs, including lost productivity resulting from metabolicdiseases-related morbidity and premature mortality.

Myostatin, also known as growth differentiation factor 8 or GDF-8, is amember of the transforming growth factor-β (TGF-β) superfamily.Myostatin is produced and released by myocytes, and is a criticalautocrine/paracrine inhibitor of skeletal muscle growth (Mouisel et al.Am J Physiol Regul Integr Comp Physiol. 2014; 307(4): R444-54).Myostatin has been primarily evaluated for use in treating diseasesassociated with muscle function.

Until now, most of metabolic diseases remain poorly treated. Currenttreatments do not fully meet patient needs, and there are no effectivetreatments applicable to the large majority of the affected patientpopulation. Accordingly, there exists an unmet need for therapies forsubjects suffering from metabolic diseases.

SUMMARY OF THE INVENTION

The present disclosure encompasses the recognition that myostatin mayact as a key regulator to directly mediate function of the muscle as anendocrine organ that controls metabolism, including body composition,and regulation thereof.

According to the invention, modulation of myostatin signaling can affecta number of metabolic parameters central to the regulation of energyproduction and consumption by selectively mobilizing body's three majorenergy pools: glucose, lipids and proteins. Without wishing to be boundby theory, it is contemplated that myostatin may play a role in theprocess both as a molecular sensor of energy expenditure and as aneffector to affect metabolic normalization. The present inventionimplicates myostatin in a broader role in metabolic regulation includingnitrogen mobilization, osmoregulation, calcium metabolism, as well asacid-base and electrolyte balance.

Thus, the present invention provides methods and compositions fortreating or preventing metabolic diseases in human subjects usinganti-pro/latent myostatin inhibitors, e.g., antibodies. The presentinvention is based, at least in part, on the discovery thatadministration of a myostatin inhibitor, e.g., an antibody, or antigenbinding fragment thereof, that specifically binds to pro/latentmyostatin to subjects having a metabolic disease, e.g., spinal cordinjury (SCI), significantly improves both the physiological and thefunctional characteristics of the injured subjects. In particular, thepresent inventors have surprisingly discovered that administration of amyostatin inhibitor, e.g., an anti-pro/latent myostatin antibodysignificantly increases the metabolic rate or energy expenditure insubjects having a metabolic disease, e.g., spinal cord injury (SCI).Administration of a myostatin inhibitor, e.g., an anti-pro/latentmyostatin antibody, also significantly attenuated SCI-induced reductionin sub-lesional muscle mass and overall body mass, while at the sametime reducing the mass of undesirable adipose tissue such as white andvisceral adipose tissue. In addition, subjects who received a myostatininhibitor, e.g., an anti-pro/latent myostatin antibody treatmentexhibited a significant improvement in their locomotor function, musclestrength, as well as motor coordination and balance skills.

The present invention is further based, at least on part, on thesurprising discovery that administration of a myostatin inhibitor, e.g.,an anti-pro/latent myostatin antibody or antigen-binding fragmentthereof, not only increases bone volume in weight-bearing bone, but alsoincreases bone volume in non-weight bearing bones, e.g., the vertebraein rodents. It is well known in the art that weight-bearing activity isan important stimulus for bone mass accrual, which could potentiallyexplain increases in bone volume in weight-bearing bones afteradministration of a myostatin inhibitor. However, the surprisingincrease observed in non-weight bearing bone volume demonstrated uponadministration of the myostatin inhibitors disclosed herein furtherconfirms that myostatin inhibitors have a broader metabolic effect,i.e., the myostatin inhibitors act not only to increase bone through,for example, increased muscle stimulation, but also act as a keyregulator to increase general metabolic effects, including bone health.

Finally, the present invention provides methods for promoting improvedbody compositions, e.g., enhanced muscle-to-fat ratios. Such methods maybe effective in achieving robust weight loss in both healthy subjects,e.g., bodybuilders, or in subjects having obesity, e.g., diet-inducedobesity, metabolic syndrome, NASH, NAFLD, and/or diabetes. As comparedto dieting alone, where weight loss occurs in both fat and muscle,administration of a myostatin inhibitor disclosed herein in combinationwith a diet leads to weight loss or greater muscle-to-fat ratios, due topreferential loss of fat stores, relative to loss of the muscle.Specifically, administration of a myostatin inhibitor in combinationwith a diet, e.g., a caloric restriction diet, results in more robustweight loss due in part to the maintenance of a higher metabolic rate;improved cardiometabolic benefits (such as lipid profile, glucosemetabolism, cardiovascular risk, etc.); and higher reduction in visceralfat and other deleterious fat levels as compared to dieting, alone. Suchbeneficial effects may be further enhanced when combined with moderateexercise.

Accordingly, in one aspect, disclosed herein is a composition comprisinga myostatin inhibitor, e.g., an antibody, or antigen-binding fragmentthereof, that specifically binds pro/latent-myostatin and blocks releaseof mature myostatin, for use as a medicament in treatment or preventionof a metabolic disease in a human subject, comprising steps of:selecting a human subject suffering from, or at risk of developing, ametabolic disease; and, administering to the human subject thecomposition comprising an effective amount of the myostatin inhibitor,e.g., antibody, or antigen-binding fragment thereof. In one embodiment,the subject is a pediatric subject.

In some embodiments, the subject does not have a myopathy, optionallywherein the myopathy is a primary myopathy or a secondary myopathy. Insome embodiments, the subject is an adult human subject suffering fromgrowth hormone (GH) deficiency, optionally wherein the subjectconcurrently receives a recombinant GH therapy or a GH gene therapy.

In some embodiments, the metabolic disease is selected from the groupconsisting of type I diabetes, type II diabetes, obesity, metabolicsyndrome/pre-diabetes, cardiovascular disease, non-alcoholicsteatohepatitis (NASH), spinal cord injury (SC1), a hypo-metabolicstate, double diabetes, Cushings disease, and an obesity syndrome. Insome embodiments, the obesity is sarcopenic obesity.

In some embodiments, the hypo-metabolic state is selected from the groupconsisting of a state associated with prolonged immobilization, a stateassociated with bed-rest, a state associated with casting, a stateassociated with a stroke, a state associated with amputation, and apost-surgery state.

In some embodiments, the Cushings disease is selected from the groupconsisting of corticosteroid-induced Cushings disease and tumor-inducedCushings disease.

In some embodiments, the obesity syndrome is selected from the groupconsisting of Prader Willi, an obesity syndrome associated with agenetic disorder, and an obesity syndrome associated with a hypothalamicdisorder.

In some embodiments, administration of the composition causes at leastone, e.g., 2, 3, 4, 5, 6, 7, 2-4, 2-5, 2-6, 3-5 or 3-6, of thefollowing:

a) increases mass and/or function of a muscle tissue in the humansubject;

b) increases mass and/or function of a fast twitch muscle tissue in thehuman subject;

c) increases mass and/or function of a slow twitch muscle tissue in thehuman subject;

d) increases the metabolic rate of the human subject;

e) increases insulin sensitivity in the human subject;

f) increases the level of brown adipose tissue in the human subject;

g) increases the level of beige adipose tissue in the human subject;

h) decreases the level of white adipose tissue in the human subject;

i) decreases the level of visceral adipose tissue in the human subject;

j) decreases the ratio of adipose-to-muscle tissue in the human subject;

k) increases glucose uptake by a target tissue in the human subject,wherein the target tissue is selected from the group consisting of brownadipose tissue, beige adipose tissue, and muscle tissue;

l) decreases glucose uptake by a target tissue in the human subject,wherein the target tissue is selected from the group consisting of awhite adipose tissue and a liver tissue;

m) decreases muscle catabolism of protein and/or muscle release of aminoacids in the human subject;

n) increases insulin dependent glycemic control in the human subject;

o) decreases intramuscular fat infiltration in the human subject;

p) improves a standardized quality of life test score;

q) prevents muscle loss or atrophy in the human subject;

r) reduces bone loss;

s) increases crossectional bone area and/or cortical thickness;

t) reduces frequency or severity of hone fractures; and/or,

u) reduces fluid overload or edema in chronic heart failure (CHF).

In some embodiments, the antibody, or antigen-binding fragment thereofdoes not bind to GDF11 or Activin. In some embodiments, the antibody, orantigen-binding fragment thereof does not bind mature (fully processed,free and active) myostatin. In some embodiments, the antibody, orantigen binding fragment thereof, comprises

a) a heavy chain variable region comprising an amino acid sequence ofSEQ ID NO:25 and a light chain variable region comprising an amino acidsequence of SEQ ID NO:31; or

b) a heavy chain comprising an amino acid sequence of SEQ ID NO:50 and alight chain comprising an amino acid sequence of SEQ ID NO:51.

In another aspect, the disclosure provides a method for treating orpreventing a metabolic disease in a human subject, the method comprisingsteps of: selecting a human subject suffering from or at risk ofdeveloping a metabolic disease; and, administering to the human subjecta composition comprising an effective amount of a myostatin inhibitor,e.g., an antibody, or antigen-binding fragment thereof, thatspecifically binds pro/latent-myostatin and blocks release of maturemyostatin, thereby treating or preventing the metabolic disease in thehuman subject.

In one embodiment, the subject does not have a myopathy. In oneembodiment, the myopathy is a primary myopathy or a secondary myopathy.

In one embodiment, the subject is an adult human subject suffering fromgrowth hormone (GH) deficiency. In one embodiment, the subjectconcurrently receives a recombinant GH therapy or a GH gene therapy.

In one embodiment, the metabolic disease is selected from the groupconsisting of type I diabetes, type 11 diabetes, obesity, metabolicsyndrome/pre-diabetes, cardiovascular disease, non-alcoholicsteatohepatitis (NASH), spinal cord injury (SCI), a hypo-metabolicstate, double diabetes, Cushings disease, and an obesity syndrome. Inone embodiment, the obesity is sarcopenic obesity. In one embodiment,the hypo-metabolic state is selected from the group consisting of astate associated with prolonged immobilization, a state associated withbed-rest, a state associated with casting, a state associated with astroke, a state associated with amputation, and a post-surgery state. Inone embodiment, the Cushings disease is selected from the groupconsisting of corticosteroid-induced Cushings disease and tumor-inducedCushings disease. In one embodiment, the obesity syndrome is selectedfrom the group consisting of Prader Willi, an obesity syndromeassociated with a genetic disorder, and an obesity syndrome associatedwith a hypothalamic disorder.

In some embodiments, the hypo-metabolic state is a post-surgery state,e.g., paraspinal muscle atrophy after lumbar spine surgery. In oneembodiment, the paraspinal muscle atrophy is a nerve injury-dependentmuscle atrophy. In one embodiment, the surgery is a spinal surgery. Inone embodiment, the spinal surgery is a lumbar spine surgery or a lumbarspine procedure, e.g., a lumbar fusion procedure, a lumbar nonfusionprocedure, a posterior lumbar fusion procedure, an anterior lumbarfusion procedure, a minimally invasive (MIS) posterior lumbardecompression procedure, a minimally invasive (MIS) posterior lumbarfusion procedure, a non-MIS equivalent procedure, etc.

In one embodiment, administration of the composition increases massand/or function of a muscle tissue in the human subject. In oneembodiment, administration of the composition increases mass and/orfunction of a fast twitch muscle tissue in the human subject. In oneembodiment, administration of the composition increases mass and/orfunction of a slow twitch muscle tissue in the human subject. In oneembodiment, administration of the composition increases the metabolicrate of the human subject. In one embodiment, administration of thecomposition increases insulin sensitivity in the human subject. In oneembodiment, administration of the composition increases the level ofbrown adipose tissue in the human subject. In one embodiment,administration of the composition increases the level of beige adiposetissue in the human subject. In one embodiment, administration of thecomposition decreases the level of white adipose tissue in the humansubject. In one embodiment, administration of the composition decreasesthe level of visceral adipose tissue in the human subject. In oneembodiment, administration of the composition decreases the ratio ofadipose-to-muscle tissue in the human subject. In one embodiment, thehuman subject is a pediatric human subject.

In one embodiment, administration of the composition increases glucoseuptake by a target tissue in the human subject, wherein the targettissue is selected from the group consisting of brown adipose tissue,beige adipose tissue, and muscle tissue. In one embodiment,administration of the composition decreases glucose uptake by a targettissue in the human subject, wherein the target tissue is selected fromthe group consisting of a white adipose tissue and a liver tissue. Inone embodiment, administration of the composition decreases musclecatabolism of protein and/or muscle release of amino acids in the humansubject. In one embodiment, the human subject is a pediatric humansubject.

In one embodiment, administration of the composition increases insulindependent glycemic control in the human subject. In one embodiment,administration of the composition decreases intramuscular fatinfiltration in the human subject. In one embodiment, administration ofthe composition achieves a clinically meaningful improvement in aquality of life score as assessed by a standardized quality of lifetest. In some embodiments, the clinically meaningful improvement is atleast an 8 point increase in the SF-36 Quality of Life Scoring System.In one embodiment, administration of the composition prevents muscleloss or atrophy in the human subject. In one embodiment, the humansubject is a pediatric human subject.

In one aspect, disclosed herein is a method for inhibiting myostatinactivation in a subject, the method comprising a step of administeringto the subject a composition comprising a myostatin inhibitor, e.g., anantibody, or antigen binding fragment thereof, that specifically bindspro/latent-myostatin and blocks release of mature myostatin, in anamount effective to cause two or more of the following in the subject:(a) an increase in mass and/or function of a muscle tissue in thesubject; (b) an increase in the metabolic rate of the subject; (c) anincrease in insulin sensitivity of the subject; (d) an increase in alevel of brown adipose tissue in the subject; (e) an increase in a levelof beige adipose tissue in the subject; (f) a decrease in a level ofwhite adipose tissue in the subject; (g) a decrease in a level ofvisceral adipose tissue in the subject; (h) a decrease in ratio ofadipose-to-muscle tissue in the subject; (i) an increase in glucoseuptake by a brown adipose tissue, a beige adipose tissue, or a muscletissue in the subject; (j) a decrease in glucose uptake by a whiteadipose tissue or a liver tissue; (k) a decrease in muscle catabolism ofprotein and/or muscle release of amino acids in the subject; (1) anincrease in insulin dependent glycemic control in the subject; (m) adecrease in intramuscular fat infiltration in the subject; (n) aclinically meaningful improvement in a quality of life score as assessedby a standardized quality of life test (e.g., at least 8 points increasein SF-36 Quality of Life Scoring System); (o) prevention of muscle lossor atrophy in the subject; and/or, (p) prevention of developing ametabolic dysregulation associated with muscle dysfunction in thesubject, wherein the subject is a human subject that benefits fromreduced myostatin signaling. In one embodiment, the human subject is apediatric human subject.

In one embodiment, the method further comprises a step of selecting thesubject suffering from a muscle condition or disorder. In anotherembodiment, the method further comprises a step of selecting the subjectsuffering from, or at risk of developing, a metabolic disorder. In oneembodiment, the method further comprises a step of selecting a pediatrichuman subject.

In one embodiment, the subject exhibits i) an increase in a level ofproMyostatin in a target muscle, as compared to a control level ofproMyostatin, or ii) a decrease in a level of latent myostatin in thecirculation, as compared to a control level of latent myostatin. In oneembodiment, the subject exhibits both i) and ii). In one embodiment, thehuman subject is a pediatric human subject.

In one embodiment, the subject has a muscle condition selected from thegroup consisting of: myopathy, muscular atrophy, muscular dystrophy,nerve injury. In one embodiment, the muscular atrophy is associated witha defect in motor neurons. In one embodiment, the defect comprises agenetic mutation. In another embodiment, the muscular atrophy isassociated with spinal muscular atrophy (SMA), amyotrophic lateralsclerosis (ALS), or myasthenia gravis. In one embodiment, the nerveinjury comprises partial denervation of neurons that innervate muscle,or impaired signaling between a motor neuron and a target muscle. In oneembodiment, the nerve injury is SCI. In another embodiment, the SCI ispartial/incomplete SCI. In one embodiment, the SCI in human subjectscomprises a lesion between i) T1-T6; ii) T7-L5; iii) C6-C7; iv) C5-C6;or v) C3-C8. In one embodiment, the subject is in an acute phase of SCI;sub-acute phase of SCI, or chronic phase of SCI. In one embodiment, thesubject has, or at risk of developing, a metabolic disorder associatedwith the SCI. In one embodiment, the metabolic disorder is or comprisesinsulin resistance, inflammation, abnormal lipid metabolism, or anincrease in intramuscular fat infiltration. In one embodiment, themuscle atrophy comprises glucocorticoid-induced muscle atrophy. In oneembodiment, the human subject is a pediatric human subject.

In one embodiment, the subject has a metabolic disease selected from thegroup consisting of type I diabetes, type 11 diabetes, obesity,metabolic syndrome/pre-diabetes, cardiovascular disease, non-alcoholicsteatohepatitis (NASH), spinal cord injury (SCI), a hypo-metabolicstate, double diabetes, Cushings disease, and an obesity syndrome. Inone embodiment, the human subject is a pediatric human subject.

In one embodiment, the subject is treated with a second therapy. In oneembodiment, the second therapy comprises neuroprotective therapy. Inanother embodiment, the neuroprotective therapy comprises a stem celltherapy.

In another aspect, disclosed herein is a method of treating orpreventing a disease associated with an impaired neurological signalingbetween a neuron and a target tissue in a human subject, the methodcomprising selecting the human subject suffering from a diseaseassociated with an impaired neurological signaling between a neuron anda target tissue; and administering to the human subject a compositioncomprising a myostatin inhibitor, e.g., an antibody, or antigen bindingfragment thereof, that specifically binds pro/latent-myostatin andblocks release of mature myostatin in an amount effective to treat orprevent the disease, thereby treating or preventing the diseaseassociated with the impaired neurological signaling in the humansubject. In one embodiment, the human subject is a pediatric humansubject.

In some embodiments, the target tissue expresses myostatin (e.g.,myostatin precursors, and/or mature myostatin). In one embodiment, thetarget tissue is selected from the group consisting of a muscle, anadipose tissue, a brain tissue, a liver tissue, and a blood vesseltissue. In one embodiment, the target tissue is a muscle.

In another aspect, disclosed herein is a method for treating a lesionthat causes an impaired but not complete loss of signaling between aneuron and a target muscle in a subject. Such method includes a step ofadministering to the subject a composition comprising a myostatininhibitor, e.g., an anti-pro/latent myostatin antibody, in an amounteffective to treat the muscle located below the lesion in the subject.In some embodiments, the amount is an amount effective to prevent muscleloss or muscle atrophy below the lesion in the subject. In someembodiments, the amount is an amount effective to increase muscle massand/or function below the lesion in the subject.

In some embodiments, the lesion is associated with incomplete spinalcord injury.

In one embodiment, the muscle contains fast-twitch muscle fibers. Inanother embodiment, the muscle located below the lesion is selected fromthe group of a soleus muscle, a gastrocnemius muscle, a bicep muscle anda tricep muscle. In one embodiment, the amount is effective to increasemass and/or function of a muscle above the lesion in the subject. Inanother embodiment, the myostatin inhibitor is an agent that blocks,antagonizes or inhibits myostatin signaling in vivo. In someembodiments, such agent is an antibody, or antigen-binding portionthereof, a small molecule, or gene therapy. In some embodiments, theantibody is an antibody that specifically binds pro/latent myostatin andblocks release of mature myostatin in vivo. In some embodiments, theantibody hinds mature myostatin. In some embodiments, the antibodyselectively (e.g., preferentially) binds mature myostatin over matureGDF11. In some embodiments, the antibody specifically binds maturemyostatin but does not bind mature GDF11. In some embodiments, theantibody binds and/or blocks a myostatin receptor.

In one embodiment, the subject has an incomplete spinal cord injury(SCI). In one embodiment, the incomplete SCI in human subjects comprisesa lesion between: i) T1-T6; ii) T7-L5; iii) C6-C7; iv) C5-C6; or v)C3-C8.

In one embodiment, the amount is effective to treat a metaboliccondition in the subject. In one embodiment, the amount is effective tocause in the subject: (a) an increase in mass and/or function of amuscle tissue in the subject; (b) an increase in the metabolic rate ofthe subject; (c) an increase in insulin sensitivity of the subject; (d)an increase in a level of brown adipose tissue in the subject; (e) anincrease in a level of beige adipose tissue in the subject; (f) adecrease in a level of white adipose tissue in the subject; (g) adecrease in a level of visceral adipose tissue in the subject; (h) adecrease in ratio of adipose-to-muscle tissue in the subject; (i) anincrease in glucose uptake by a brown adipose tissue, a beige adiposetissue, or a muscle tissue in the subject; (j) a decrease in glucoseuptake by a white adipose tissue or a liver tissue; (k) a decrease inmuscle catabolism of protein and/or muscle release of amino acids in thesubject; (1) an increase in insulin dependent glycemic control in thesubject; (m) a decrease in intramuscular fat infiltration in thesubject; (n) at least 8 points increase in SF-36 Quality of Life ScoringSystem; (o) prevention of muscle loss or atrophy in the subject; and/or,(p) prevention of developing a metabolic dysregulation associated withmuscle dysfunction in the subject. In one embodiment, the subject is apediatric subject.

In another aspect, the disclosure provides a method of treating orpreventing a metabolic disease in a human subject, the method comprisingselecting a human subject suffering from a metabolic disease; andadministering to the human subject an effective amount of an antibody,or antigen binding fragment thereof, that specifically bindspro/latent-myostatin, thereby treating or preventing the metabolicdisease in the human subject. In one embodiment, the human subject is apediatric human subject.

In one embodiment, the metabolic disease is selected from the groupconsisting of type I diabetes, type II diabetes, obesity, metabolicsyndrome/pre-diabetes, cardiovascular disease, non-alcoholicsteatohepatitis (NASH), spinal cord injury (SCI), a hypo-metabolicstate, double diabetes, Cushings disease, and an obesity syndrome. Inone embodiment, the obesity is sarcopenic obesity. In one embodiment,the hypo-metabolic state is selected from the group consisting of astate associated with prolonged immobilization, a state associated withbed-rest, a state associated with casting, a state associated with astroke, a state associated with amputation, and a post-surgery state. Inone embodiment, the Cushings disease is selected from the groupconsisting of corticosteroid-induced Cushings disease and tumor-inducedCushings disease. In one embodiment, the obesity syndrome is selectedfrom the group consisting of Prader Willi, an obesity syndromeassociated with a genetic disorder, and an obesity syndrome associatedwith a hypothalamic disorder.

In another aspect, the disclosure provides a method of treating orpreventing a disease associated with an impaired neurological signalingbetween a neuron and a target tissue in a human subject, the methodcomprising selecting a human subject suffering from a disease associatedwith an impaired neurological signaling between a neuron and a targettissue; and administering to the human subject an effective amount of amyostatin inhibitor, e.g., an antibody, or antigen binding fragmentthereof, that specifically binds pro/latent-myostatin, thereby treatingor preventing the disease associated with the impaired neurologicalsignaling in the human subject. In some embodiments, the target tissueexpresses myostatin (e.g., myostatin precursors, and/or maturemyostatin). In one embodiment, the human subject is a pediatric humansubject.

In one embodiment, the disease associated with an impaired neurologicalsignaling between a neuron and a target tissue is selected from thegroup consisting of spinal cord injury (SCI), myasthenia gravis,amyotrophic lateral sclerosis (ALS), and spinal muscular atrophy (SMA).In one embodiment, the disease associated with an impaired neurologicalsignaling between a neuron and a target tissue is spinal cord injury(SCI). In one embodiment, the human subject is in an acute spinal cordinjury (SCI) phase. In one embodiment, the human subject is in asub-acute spinal cord injury (SCI) phase. In one embodiment, the humansubject is in a chronic spinal cord injury (SCI) phase.

In one embodiment, the target tissue is selected from the groupconsisting of a muscle tissue, an adipose tissue, a brain tissue, aliver tissue, and a blood vessel tissue.

In one embodiment, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, increases mass and/orfunction of a muscle tissue in the human subject. In one embodiment,administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, increases mass and/or function of a fasttwitch muscle tissue in the human subject. In one embodiment,administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, increases mass and/or function of a slowtwitch muscle tissue in the human subject. In one embodiment, the humansubject is a pediatric human subject.

In one embodiment, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, increases the metabolicrate of the human subject. In one embodiment, administration of themyostatin inhibitor, e.g., antibody, or antigen binding fragmentthereof, increases insulin sensitivity in the human subject. In oneembodiment, administration of the myostatin inhibitor, e.g., antibody,or antigen binding fragment thereof, increases the level of brownadipose tissue in the human subject. In one embodiment, administrationof the myostatin inhibitor, e.g., antibody, or antigen binding fragmentthereof, increases the level of beige adipose tissue in the humansubject. In one embodiment, administration of the myostatin inhibitor,e.g., antibody, or antigen binding fragment thereof, decreases the levelof white adipose tissue in the human subject. In one embodiment,administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, decreases the level of visceral adipose tissuein the human subject. In one embodiment, administration of the myostatininhibitor, e.g., antibody, or antigen binding fragment thereof,decreases the ratio of adipose-to-muscle tissue in the human subject. Inone embodiment, the human subject is a pediatric human subject.

In one embodiment, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, increases glucose uptakeby a muscle tissue in the human subject. In one embodiment,administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, decreases glucose uptake by a target tissue,wherein the target tissue is selected from the group consisting of awhite adipose tissue, a liver tissue and a blood vessel tissue. In oneembodiment, the human subject is a pediatric human subject.

In one embodiment, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, decreases musclecatabolism of protein and/or muscle release of amino acids in the humansubject. In one embodiment, administration of the myostatin inhibitor,e.g., antibody, or antigen binding fragment thereof, increases insulindependent glycemic control in the human subject. In one embodiment, thehuman subject is a pediatric human subject.

In another aspect, disclosed herein is a method of increasing metabolicrate in a human subject, the method comprising selecting a human subjectwho would benefit from an increase in metabolic rate; and administeringto the human subject an effective amount of a myostatin inhibitor, e.g.,an antibody, or antigen binding fragment thereof, that specificallybinds pro/latent-myostatin, thereby increasing the metabolic rate in thehuman subject.

In another aspect, disclosed herein is a method of increasing the levelof brown adipose tissue in a human subject, the method comprisingselecting a human subject who would benefit from an increase in thelevel of brown adipose tissue; and administering to the human subject aneffective amount of a myostatin inhibitor, e.g., an antibody, or antigenbinding fragment thereof, that specifically binds pro/latent-myostatin,thereby increasing the level of brown adipose tissue in the humansubject.

In another aspect, disclosed herein is a method of increasing the levelof beige adipose tissue in a human subject, the method comprisingselecting a human subject who would benefit from an increase in thelevel of beige adipose tissue; and administering to the human subject aneffective amount of a myostatin inhibitor, e.g., an antibody, or antigenbinding fragment thereof, that specifically binds pro/latent-myostatin,thereby increasing the level of beige adipose tissue in the humansubject.

In another aspect, disclosed herein is a method of increasing insulindependent glycemic control in a human subject, the method comprisingselecting a human subject who would benefit from an increase in insulindependent glycemic control; and administering to the human subject aneffective amount of a myostatin inhibitor, e.g., an antibody, or antigenbinding fragment thereof, that specifically binds pro/latent-myostatin,thereby increasing insulin dependent glycemic control in the humansubject.

In another aspect, disclosed herein is a method of decreasing musclecatabolism of protein and/or muscle release of amino acids in a humansubject, the method comprising selecting a human subject who wouldbenefit from a decrease in muscle catabolism of protein and/or musclerelease of amino acids; and administering to the human subject aneffective amount of a myostatin inhibitor, e.g., an antibody, or antigenbinding fragment thereof, that specifically binds pro/latent-myostatin,thereby decreasing muscle catabolism of protein and/or muscle release ofamino acids in the human subject.

In another aspect, disclosed herein is a method of decreasing glucoseuptake by a target tissue in a human subject, the method comprisingselecting a human subject who would benefit from a decrease in glucoseuptake by a target tissue selected from the group consisting of a whiteadipose tissue, a liver tissue and a blood vessel tissue; andadministering to the human subject an effective amount of a myostatininhibitor, e.g., an antibody, or antigen binding fragment thereof, thatspecifically binds pro/latent-myostatin, thereby decreasing glucoseuptake by the target tissue in the human subject.

In one embodiment, the target tissue comprises macrophages, smoothmuscle cells and foam cells.

In another aspect, disclosed herein is a method of treating orpreventing a metabolic disease in a human subject, the method comprisingselecting a human subject suffering from a metabolic disease; andadministering to the human subject an amount of a myostatin inhibitor,e.g., an antibody, or antigen binding fragment thereof, thatspecifically binds pro/latent-myostatin, effective to cause at least twoor more of the following in the human subject: (a) an increase in massand/or function of a muscle tissue in the human subject; (b) an increasein the metabolic rate of the human subject; (c) an increase in insulinsensitivity of the human subject; (d) an increase in the level of brownadipose tissue in the human subject; (e) an increase in the level ofbeige adipose tissue in the human subject; (f) a decrease in the levelof white adipose tissue in the human subject; (g) a decrease in thelevel of visceral adipose tissue in the human subject; (h) a decrease inthe ratio of adipose-to-muscle tissue in the human subject; (i) anincrease in glucose uptake by a white adipose tissue, a liver tissue ora blood vessel tissue in the human subject; (j) a decrease in musclecatabolism of protein and/or muscle release of amino acids in the humansubject; and/or (k) an increase in insulin dependent glycemic control inthe human subject, thereby treating or preventing the metabolic diseasein the human subject. In one embodiment, the human subject is apediatric human subject.

In another aspect, disclosed herein is a method of increasing massand/or function of a muscle located below a lesion in a subject who hassuffered a lesion, the method comprising selecting a subject who hassuffered a lesion; and administering to the human subject an effectiveamount of a myostatin inhibitor, e.g., an antibody, or antigen bindingfragment thereof, that specifically hinds pro/latent-myostatin, therebyincreasing the mass and/or function of the muscle located below a lesionin the human subject.

In one embodiment, the lesion is due to a spinal cord injury (SCI). Inone embodiment, the human subject is in an acute spinal cord injury(SCI) phase. In one embodiment, the human subject is in a sub-acutespinal cord injury (SCI) phase. In one embodiment, the human subject isin a chronic spinal cord injury (SCI) phase.

In one embodiment, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, further increases massand/or function of a muscle above the lesion. In one embodiment,administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, increases mass and/or function of a fastswitch muscle. In one embodiment, administration of the antibody, orantigen binding fragment thereof, increases mass and/or function of aslow switch muscle.

In some embodiments, the mass of the muscle tissue is increased by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, themass of the muscle tissue is increased by at least about 1-5%, 5-10%,10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or50-100%.

In some embodiments, the function of the muscle tissue is increased byat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments,the function of the muscle tissue is increased by at least about 1-5%,5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%,40-90%, or 50-100%.

In one embodiment, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, increases locomotorfunction in the human subject. In some embodiments, the locomotorfunction of the human subject is increased by at least 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,70%, 80%, 90% or 100%. In other embodiments, the locomotor function ofthe human subject is increased by at least about 1-5%, 5-10%, 10-20%,1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.In one embodiment, the human subject is a pediatric human subject.

In one embodiment, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, increases motorcoordination and balance in the human subject. In some embodiments, themotor ordination and balance of the human subject is increased by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, themotor ordination and balance of the human subject is increased by atleast about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%,20-60%, 30-80%, 40-90%, or 50-100%. In one embodiment, the human subjectis a pediatric human subject.

In one embodiment, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, increases muscle strengthin the human subject. In some embodiments, the muscle strength of thehuman subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or100%. In other embodiments, the muscle strength of the human subject isincreased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%,10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. In one embodiment,the human subject is a pediatric human subject.

In one embodiment, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, increases grip strengthin the human subject. In one embodiment, administration of the myostatininhibitor, e.g., antibody, or antigen binding fragment thereof,decreases the level of white adipose tissue in the human subject. Insome embodiments, the level of white adipose tissue is decreased by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, thelevel of white adipose tissue is decreased by at least about 1-5%,5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%,40-90%, or 50-100%. In one embodiment, the human subject is a pediatrichuman subject.

In one embodiment, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, increases total body massof the human subject. In some embodiments, the level of total body massis increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In otherembodiments, the level of total body mass is increased by at least about1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%,30-80%, 40-90%, or 50-100%. In one embodiment, the human subject is apediatric human subject.

In one embodiment, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, increases metabolic rateof the human subject. In some embodiments, the metabolic rate isincreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In otherembodiments, the metabolic rate is increased by at least about 1-5%,5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%,40-90%, or 50-100%. In one embodiment, the human subject is a pediatrichuman subject.

In one embodiment, the muscle is selected from the group of a soleusmuscle, a gastrocnemius muscle, a bicep muscle and a tricep muscle.

In one embodiment, the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, is administered to the human subject withinless than 5, 10, 20, 30, 40, 50, 60 minutes after the human subject hassuffered the lesion. In one embodiment, the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, is administered to thehuman subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 or 24 hoursafter the human subject has suffered the lesion. In one embodiment, themyostatin inhibitor, e.g., antibody, or antigen binding fragmentthereof, is administered to the human subject within at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10 days or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 24, 48 or 60 months after the human subject has suffered the lesion.In one embodiment, the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, is administered to the human subject for about1-30 days, about 1-50 days, about 1-100 days, about 1-200 days or about1-300 days.

In one embodiment, the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, is administered to the human subjectchronically. In one embodiment, the myostatin inhibitor, e.g., antibody,or antigen binding fragment thereof, is administered to the humansubject at a dose in a range of 0.01 mg/kg to 100 mg/kg. In oneembodiment, the myostatin inhibitor, e.g., antibody, or antigen bindingfragment thereof, is administered to the human subjectintraperitoneally, intravenously, intramuscularly, locally orsubcutaneously.

In one embodiment, the methods disclosed herein further compriseadministering a second therapy to the human subject. In one embodiment,the second therapy is selected from the group consisting of insulin,insulin sensitivity enhancing agents, alpha-glucosidase inhibitors,biguanides, sulfonylureas, insulin secretion-promoting agents, amyrinagonist, phosphotyrosin phosphatase inhibitor, aldose reductaseinhibitors, neurotrophic factors, PKC inhibitors, advanced glycationend-product (AGE) inhibitors, active oxygen quenching agents, statins,squalene synthetase inhibitors, fibrate, niacin, PCSK9 inhibitors,triglyceride lowing agents, cholesterol sequestering agents, angiotensinconverting enzyme inhibitors, angiotensin II antagonists, calciumchannel blockers, ursodiol, pioglitazone, orlistat, betaine,rosiglitazone, central anti-obesity agents, gastrointestinal lipaseinhibitors, beta 3-adrenoceptor agonists, peptide-basedappetite-suppressing agents, cholecystokinin agonists, dopamineagonists, DPP-4 inhibitors, glucagon-like peptides, meglitinides,sulfonylureas, sodium glucose transporter (SGLT) 2 inhibitors,cyclooxygenase inhibitors, progesterone derivatives,metoclopramide-based agents, tetrahydrocannabinol-based agents, andlipid metabolism improving agents.

In one embodiment, the myostatin inhibitor, e.g., antibody, orantigen-binding fragment thereof, is administered at a dose of about0.01 mg/kg to about 30 mg/kg. In one embodiment, the myostatininhibitor, e.g., antibody, or antigen-binding fragment thereof, isadministered intraperitoneally, intravenously, intramuscularly, orsubcutaneously.

In one embodiment, the antibody, or antigen-binding fragment thereofdoes not bind to GDF11 or Activin. In one embodiment, the antibody, orantigen-binding fragment thereof does not bind mature myostatin. In oneembodiment, the antibody, or antigen binding fragment thereof, iscross-reactive with human and murine pro/latent myostatin. In oneembodiment, the antibody, or antigen binding fragment thereof, inhibitsproteolytic formation of mature myostatin by tolloid protease. In oneembodiment, the antibody, or antigen binding fragment thereof, inhibitsproteolytic formation of mature myostatin by tolloid protease with anIC50 of less than 1 μM.

In one embodiment, the antibody, or antigen binding fragment thereof,comprises a heavy chain variable domain comprising a complementaritydetermining region 3 (CDRH3) comprising a sequence as set forth in anyone of SEQ ID NOs:10-11 and 66. In one embodiment, the antibody, orantigen binding fragment thereof, comprises a light chain variabledomain comprising a complementarity determining region 3 (CDRL3)comprising a sequence as set forth in any one of SEQ ID NO: 22-23 and67. In one embodiment, the antibody, or antigen binding fragmentthereof, comprises six complementarity determining regions (CDRs):CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein CDRH1 comprises asequence as set forth in any one of SEQ ID NOs: 1-3, CDRH2 comprises asequence as set forth in any one of SEQ ID NOs: 4-9, CDRH3 comprises asequence as set forth in any one of SEQ ID NOs: 10-11 and 66, CDRL1comprises a sequence as set forth in any one of SEQ ID NOs: 12-17, CDRL2comprises a sequence as set forth in any one of SEQ ID NOs: 18-21, andCDRL3 comprises a sequence as set forth in any one of SEQ ID NOs: 22-23and 67.

In one embodiment, CDRH1 comprises a sequence as set forth in SEQ ID NO:1 or 2, CDRH2 comprises a sequence as set forth in SEQ ID NO: 4 or 5,CDRH3 comprises a sequence as set forth in SEQ ID NO: 10, CDRL1comprises a sequence as set forth in SEQ ID NO: 12 or 13, CDRL2comprises a sequence as set forth in SEQ ID NO: 18 or 19, and CDRL3comprises a sequence as set forth in SEQ ID NO: 22.

In one embodiment, CDRH1 comprises a sequence as set forth in SEQ ID NO:1 or 3, CDRH2 comprises a sequence as set forth in SEQ ID NO: 6 or 7,CDRH3 comprises a sequence as set forth in SEQ ID NO: 11, CDRL1comprises a sequence as set forth in SEQ ID NO: 14 or 15, CDRL2comprises a sequence as set forth in SEQ ID NO: 20 or 21, and CDRL3comprises a sequence as set forth in SEQ ID NO: 23.

In one embodiment, CDRH1 comprises a sequence as set forth in SEQ ID NO:1 or 2, CDRH2 comprises a sequence as set forth in SEQ ID NO: 4 or 5,CDRH3 comprises a sequence as set forth in SEQ ID NO: 66, CDRL1comprises a sequence as set forth in SEQ ID NO: 12 or 13, CDRL2comprises a sequence as set forth in SEQ ID NO: 18 or 19, and CDRL3comprises a sequence as set forth in SEQ ID NO: 67.

In one embodiment, CDRH1 comprises a sequence as set forth in SEQ ID NO:1 or 3, CDRH2 comprises a sequence as set forth in SEQ ID NO: 8 or 9,CDRH3 comprises a sequence as set forth in SEQ ID NO: 11, CDRL1comprises a sequence as set forth in SEQ ID NO: 16 or 17, CDRL2comprises a sequence as set forth in SEQ ID NO: 20 or 21, and CDRL3comprises a sequence as set forth in SEQ ID NO: 23.

In one embodiment, the antibody, or antigen binding fragment thereof,wherein the antibody comprises a heavy chain variable domain sequence asset forth in any one of SEQ ID NOs: 24-29. In one embodiment, theantibody, or antigen binding fragment thereof, comprises a light chainvariable domain sequence of as set forth in any one of SEQ ID NOs:30-35. In one embodiment, the antibody, or antigen binding fragmentthereof, comprises a heavy chain variable region comprising an aminoacid sequence of SEQ ID NO:25 and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO:31.

In one embodiment, the antibody, or antigen binding fragment thereof,comprises a heavy chain comprising an amino acid sequence of SEQ IDNO:50. In one embodiment, the antibody, or antigen binding fragmentthereof, comprises a light chain comprising an amino acid sequence ofSEQ ID NO:51.

In one embodiment, the antibody, or antigen binding fragment thereof,competes for binding to pro/latent myostatin with any other antibodydescribed herein. In one embodiment, the antibody, or antigen bindingfragment thereof, binds to pro/latent myostatin at the same epitope asan antibody described herein.

In one embodiment, the antibody, or antigen binding fragment thereof,competes for binding to pro/latent myostatin with an equilibriumdissociation constant, Kd, between the antibody and pro/latent myostatinof less than 10⁻⁶ M. In one embodiment, the Kd is in a range of 10⁻¹¹ Mto 10⁻⁶ M.

In one embodiment, the antibody, or antigen binding fragment thereof, isa human antibody, a humanized antibody, a diabody, a chimeric antibody,a Fab fragment, a F(ab′)2 fragment, or an Fv fragment. In oneembodiment, the antibody is a humanized antibody. In one embodiment, theantibody is a human antibody. In one embodiment, the antibody, orantigen binding fragment thereof, comprises a framework having a humangermline sequence.

In one embodiment, the antibody, or antigen binding fragment thereof,comprises a heavy chain constant domain selected from the groupconsisting of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1,IgA2, IgD, IgM, and IgE constant domains. In one embodiment, theantibody comprises a constant domain of IgG4. In one embodiment, theantibody comprises a constant domain of IgG4 having a backbonesubstitution of Ser to Pro that produces an IgG1-like hinge and permitsformation of inter-chain disulfide bonds. In one embodiment, theantibody, or antigen-binding portion thereof, docs not bind to GDF11. Inone embodiment, the antibody, or antigen-binding portion thereof, doesnot bind mature (fully processed, free/soluble, active) myostatin. Inone embodiment, the antibody, or antigen-binding portion thereof,selectively or preferentially binds the tissue-bound myostatin (e.g.,pro-form of myostatin; i.e., proMyostatin or pro-myostatin). In oneembodiment, the antibody, or antigen-binding portion thereof, binds boththe pro- and latent forms of myostatin (proMyostatin and latentMyostatin) but not mature myostatin.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B depict myostatin (also known as GDF8) domain structure andpro-myostatin assembly. FIG. 1A shows myostatin secreted as aproprotein, with an inhibitory prodomain followed by a C-terminal growthfactor domain, which exists as a disulfide-linked dimer. FIG. 1B showsprecursor protein assembled in an inactive conformation where theprodomain (dark gray) encloses the growth factor (light gray) with a“straightjacket” assembly. This figure is an adaption from the structureof latent TGFβ1 (Shi et al. Nature 2011).

FIG. 2 demonstrates that the activation of myostatin involves twodistinct protease events, generating three major myostatin species. Thebiosynthetic precursor protein, pro-myostatin, is processed by twoseparate proteases. Cleavage of pro-myostatin (and pro-GDF11) is carriedout by a proprotein convertase, such as Furin/PACE3 (Paired Basic Aminoacid Cleaving Enzyme 3) or PCSK5 (Proprotein Convertase Subtilisin/Kexintype 5), which cuts at a conserved RXXR site between the prodomain andmature growth factor. This cleavage produces a latent complex, in whichthe mature growth factor is shielded from binding to its receptors bythe prodomain Activation and release of the active growth factor isaccomplished after cleavage by an additional protease from theBMP/tolloid family, such as TLL-2 (Tolloid-like protein 2) or BMP1 (BoneMorphogenetic Protein 1). These cleavage events yield a mature form ofmyostatin, which may be referred to as active myostatin or maturemyostatin.

FIG. 3 depicts body mass in naïve mice, and sham, SCI-veh, SCI-IgG,SCI-Ab1 treatment groups at 1- and 2-weeks post-SCI. Asterisks * on thetop reflect significant difference from sham; and asterisks * at thebottom of the bars reflect significant difference from SCI-Ab1.

FIG. 4 depicts muscle wet weight (mass) in sham, SCI-veh, SCI-IgG,SCI-Ab1 treatment groups at 2-weeks post-SCI. Excised muscle includessublesional soleus and gastrocnemius and supralesional biceps andtriceps muscles.

FIG. 5 depicts analysis of total fat-free (lean) and fat mass in sham,SCI-veh, SCI-IgG, and SCI-Ab1 treatment groups at 2-weeks post-SCI.

FIG. 6 depicts lean mass as a percentage of body mass in sham, SCI-veh,SCI-IgG, SCI-Ab1 treatment groups at 2-weeks post-SCI.

FIG. 7 depicts analysis of kcal/hr and TEE in sham, SCI-veh, SCI-IgG,and SCI-Ab1 treatment groups at 2-weeks post-SCI. In the lower graphs,the SCI/Treatment Control group represents the combined SCI/vch+SCI/IgGgroups from the upper graphs.

FIG. 8 depicts the BMS locomotor assessment in sham, SCI-veh, SCI-IgG,and SCI-Ab1 groups, at baseline (before survival surgery), 1-day,1-week, and 2-weeks post-SCI. Statistical comparison at 1- and 2-weekspost-SCI reflect combined SCI-veh+SCI-IgG data.

FIG. 9 depicts Rotarod time scores in sham, SCI-veh, SCI-IgG, andSCI-Ab1 groups, after pre-training (PT), 1-week, and 2-weeks post-SCI.

FIG. 10 depicts grip strength in sham, SCI-veh, SCI-IgG, and SCI-Ab1groups, after pre-training (PT), 1-week, and 2-weeks post-SCI.

FIGS. 11A-11D show effects of treatment with Ab2 on change in lean massin healthy Cynomolgus monkeys. Healthy male Cynomolgus monkeys weredosed by intravenous injection once weekly for 8 weeks at threedifferent doses of Ab2, 3 mg/kg, 10 mg/kg, and 30 mg/kg, with a 4-weekrecovery phase. Control animals were administered vehicle control (20 mMCitrate and 150 mM Sodium Chloride USP, pH 5.5). Lean mass was measuredby Dual Energy X-Ray Absorptiometry (DEXA). FIG. 11A is a graph showingmean percent change in lean mass in muscles from all limbs inAb2-treated and control animals measured at Day 0, 4 weeks, 8 weeks, and12 weeks. FIG. 11B is a graph showing mean percent change in lean massin muscles from all limbs in Ab2-treated and vehicle control animalsmeasured at week 4. FIG. 11C is a graph showing mean percent change inlean mass in limb muscles in Ab2-treated and vehicle control animalsmeasured at week 8. FIG. 11D is a graph showing mean percent change inlean mass in limb muscles in Ab2-treated and vehicle control animalsmeasured at week 12.

FIGS. 12A-2B are graphs showing effects of treatment with Ab2 on muscleweight in biceps brachii and gastrocnemius muscles collected fromhealthy Cynomolgus monkeys. Healthy male Cynomolgus monkeys were dosedby intravenous injection once weekly for 8 weeks at three differentdoses of Ab2, 3 mg/kg, 10 mg/kg, and 30 mg/kg, with a 4-week recoveryphase to week 12. Control animals were administered vehicle control (20mM Citrate and 150 mM Sodium Chloride USP, pH 5.5). Muscle weight wasmeasured by tissue weight at week 12.

FIG. 13 shows mean percent change in lean mass from baseline (day 0) andin percent difference in muscle weight in healthy Cynomolgus monkeystreated with Ab2 compared to the vehicle control.

FIGS. 14A and 14B show latent Myostatin levels in serum samples ofAb2-treated healthy Cynomolgus monkeys and in control animals measuredusing quantitative fluorescent western blotting. Healthy male Cynomolgusmonkeys were dosed by intravenous injection once weekly for 8 weeks atthree different doses, 3 mg/kg, 10 mg/kg, and 30 mg/kg, with a 4-weekrecovery phase. Control animals were administered vehicle control (20 mMCitrate and 150 mM Sodium Chloride USP, pH 5.5). Serum samples werecollected over different study days and relative levels of latentMyostatin in the serum samples were analyzed using quantitativefluorescent western blotting.

FIG. 15 depicts lean mass change by Ab2-mediated Myostatin inhibition.

FIG. 16 depicts differentially expressed genes (DEGs) in Ab2-treatedgroups.

FIG. 17 depicts repression of atrogenes after Ab2-mediated myostatininhibition.

FIG. 18 depicts expression of muscle specific markers after Ab2-mediatedmyostatin inhibition.

FIG. 19 depicts expression of markers of respiratory capacity afterAb2-mediated myostatin inhibition.

FIG. 20 depicts expression of markers of adipocytes and adipogenesisafter Ab2-mediated myostatin inhibition.

FIG. 21 depicts regulation of pyruvate dehydrogenase.

FIG. 22 depicts expression levels of regulators of pyruvatedehydrogenase and fatty acid oxidation.

FIG. 23 depicts an immunofluorescence assay performed on cryosectionedtibialis anterior muscle from healthy mice using Ab2, and co-stainedwith laminin.

FIGS. 24A-24B show cross sections of tibilias anterior muscle probedwith anti-pro/latent GDF8 antibody, Ab10 or non-specific targetingantibody, is shown in FIG. 24A, HuNeg is shown in FIG. 24B, and each ofthe figures are counterstained with DAPI. The scale bar is 0.01 cm.

FIGS. 25A-25C show cross sections of tibilias anterior muscle probedwith anti-pro/latent GDF8 antibody, Ab10, that had been incubated inblocking buffer alone (FIG. 25A), incubated in blocking buffer with10-fold molar excess recombinant mouse GDF8 (FIG. 25B), or incubated inblocking buffer with 10-fold molar excess recombinant mouse GDF11 (FIG.25C). FIGS. 25A-25C are counterstained with DAPI.

FIGS. 26A-26C show cross sections of tibilias anterior muscle probedwith anti-pro/latent GDF8 antibody, Ab10, and anti-laminin, andcounterstained with DAPI. Pro/latent GDF8 and laminin colocalize in theinterstitial space at muscle fiber vertices (arrow), between musclefibers (arrow head), and around interstitial nuclei (asterisk).

FIGS. 27A-27C demonstrate reduction of SCI-induced intramuscular fatinfiltration by a monoclonal antibody that inhibits activation ofmyostatin.

FIGS. 28A-28B show effects of a monoclonal antibody that inhibitsactivation of myostatin in a cardiotoxin-induced injury model.

FIG. 29 demonstrates that antibody-treated animals showed astatistically significant increase in mean total crossectional bone areaand cortical thickness as compared to control (PBS).

FIG. 30 demonstrates that antibody-treated animals showed an increase intrabecular bone volume, trabecular thickness, and trabecular number ascompared to control. Additionally, antibody-treated animals showed adecrease in trabecular separation as compared to control.

FIG. 31 demonstrates that animals treated with the myostatin inhibitordemonstrated an increase in bone volume in non-weight bearing bone,e.g., the vertebrae.

FIG. 32 demonstrates that mice treated with Ab1 exhibited a 14.4%increase in body weight at day 50 as compared to control mice (PBStreatment).

FIG. 33 depicts the increase in weight of several muscles:gastrocnemius, TA, EDL, soleus, and masseter, after treatment with Ab1.

FIG. 34A depicts an increase of 23% in plantartlexor force (maximumtorque) after treatment with Ab1 versus PBS control, and a 20% increasein plantarflexor force maximum torque/limb length after treatment withAb1 versus PBS control. FIG. 34B depicts masseter force after treatmentwith Ab1 versus controls.

FIG. 35 depicts histology data from a high-dose SMN-C1 cohort and showsthe total fiber cross sectional area (CSA) and a histogram of CSAdistribution in control (vehicle) versus Ab1 treated animals,demonstrating an increasing trend in fiber CSA. This trend wasattributed entirely to type IIb fibers (data not shown).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery thatadministration of a myostatin inhibitor, e.g., an antibody, or antigenbinding fragment thereof, that specifically binds to pro/latentmyostatin to subjects having a metabolic disease, e.g., spinal cordinjury (SCI), significantly improves both the physiological and thefunctional characteristics of the affected subjects. In particular, thepresent inventors have surprisingly discovered that administration of amyostatin inhibitor, e.g., an anti-pro/latent myostatin antibody orantigen-binding portion thereof, significantly enhances the metabolicrate or energy expenditure in subjects having metabolic disease ordysfunction. Administration of a myostatin inhibitor, e.g., ananti-pro/latent myostatin antibody also significantly attenuatedSCI-induced reduction in sub-lcsional muscle mass and overall body massand, while at the same time reducing the mass of undesirable adiposetissue such as white and visceral adipose tissue. In addition, subjectswho received a myostatin inhibitor, e.g., an anti-pro/latent myostatinantibody or antigen-binding portion thereof, treatment exhibited asignificant improvement in their locomotor function, muscle strength, aswell as motor coordination and balance skills

Accordingly, the present invention provides methods for treating orpreventing metabolic disease in a human subject using a myostatininhibitor, e.g., anti-pro/latent myostatin antibodies or antigen-bindingportions thereof. The present invention also provides methods fortreating or preventing diseases associated with an impaired neurologicalsignaling, increasing metabolic rate, increasing the level of brownadipose tissue, increasing the level of beige adipose tissue, increasinginsulin dependent glycemic control, decreasing muscle catabolism ofprotein and/or muscle release of amino acids, decreasing glucose uptakeby a target tissue in a human subject using a myostatin inhibitor, e.g.,anti-pro/latent myostatin antibodies or antigen-binding portionsthereof. The present invention further provides methods for increasingmass and/or function of a muscle located below a lesion in a subject whohas suffered a lesion using a myostatin inhibitor, e.g., anti-pro/latentmyostatin antibodies and antigen-binding portions thereof.

Thus, the present invention includes the use of antibodies andantigen-binding portions thereof that specifically bind proMyostatinand/or latent myostatin and block activation of mature myostatin in vivoin subjects, e.g., human subjects who benefit from reduced myostatinsignaling. The invention includes methods of treating or preventingconditions associated with myostatin dysregulation using myostatininhibitors, e.g., antibodies and antigen-binding portions thereof, thatspecifically bind proMyostatin and/or latent myostatin and blockactivation of myostatin in an amount effective to treat or prevent suchconditions.

Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±1%. Furthermore, the term “about” can mean within ±1% of a value.

The terms “administer”, “administering” or “administration” include anymethod of delivery of an antibody or an antigen-binding fragmentthereof, e.g., a pharmaceutical composition comprising such an antibodyor antigen-binding fragment, or an agent, into a subject's system or toa particular region in or on a subject (systemic and localadministration, respectively).

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1 CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region is comprised of one domain, CL. The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The antibodies of theinvention are described in further detail in International PatentApplication WO2016073853A1 and International Application No.PCT/US2016/052014, filed on Sep. 15, 2016, the entire contents of eachof which are incorporated herein by reference. Antibody variants, asknown in the art, are also encompassed by the present invention.

The term “antigen binding fragment”, “antigen-binding fragment” or“antigen-binding portion” of an antibody (or simply “antibody fragment”or “antibody portion”), as used herein, refers to one or more fragmentsof an antibody that retain the ability to specifically bind to anantigen (e.g., pro/latent myostatin). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen binding fragment” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consistsof a VH domain; and (vi) an isolated complementarity determining region(CDR). Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J. et al. (1994) Structure 2:1121-1123).

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

The term “control” or “control sample,” as used herein, refers to anyclinically or scientifically relevant comparative sample or counterpart,including, for example, a sample from a healthy subject, a sample from asubject having a deficiency that can cause or make the subjectsusceptible to a certain disease or condition, a subject with a diseaseor condition of interest, a sample from a subject treated with apharmaceutical carrier, a sample from a subject prior to treatment, asham or buffer treated subject or sample, an untreated subject orsample, and the like.

The term “control level” refers to an accepted or pre-determined levelof a biological marker, e.g., a level of a marker obtained beforetreatment or the onset of disease or before administration of a drug,e.g., an antibody or an antigen-binding portion thereof. The level of abiological marker present in a subject or population of subjects havingone or more particular characteristics, e.g., the presence or absence ofa particular disease or condition.

The term “decrease”, as used herein, in the context of a disease symptomrefers to a statistically significant decrease in such level. Thedecrease can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or belowthe level of detection for the detection method. The decrease can alsobe, for example, about 1-10%, 10-20%, 1-30%, 20-50%, 30-60%, 40-70%,50-80%, or 60-90% below the level of detection for the detection method.In certain embodiments, the reduction is down to a level accepted aswithin the range of normal for an individual without such disorder whichcan also be referred to as a normalization of a level.

As used herein, the term “denervation” refers to loss or perturbation ofnerve supply or neuronal input to its target tissue, such as a muscletissue. Causes of denervation include disease (e.g., genetic disordersof motor neurons), chemical toxicity, physical injury, or intentionalsurgical interruption of a nerve and the like. Denervation may bepartial denervation (also referred to as incomplete denervation) orcomplete denervation. Partial denervation can be, for example, at least1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% loss or perturbation of nervesupply or neuronal input to its target tissue. In some embodiments,partial denervation includes about 1-10%, 10-20%, 1-30%, 20-50%, 30-60%,40-70%, 50-80%, 60-90% of loss or perturbation of nerve supply orneuronal input to its target tissue.

“Determining” as used herein is understood as performing an assay orusing a method to ascertain the state of someone or something, e.g., thepresence, absence, level, or degree of a certain condition, biomarker,disease state, or physiological condition.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques.However, development also refers to progression that may beundetectable. For purpose of this disclosure, development or progressionrefers to the biological course of the symptoms. “Development” includesoccurrence, recurrence, and onset. As used herein “onset” or“occurrence” of a disease/disorder associated with myopathy includesinitial onset and/or recurrence.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The abbreviation,“e.g.” is derived from the Latin exempli gratia, and is used herein toindicate a non-limiting example. Thus, the abbreviation “e.g.” issynonymous with the term “for example.”

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. In certain embodiments, an antibody is said to specificallybind an antigen when it preferentially recognizes its target antigen ina complex mixture of proteins and/or macromolecules. The epitope can bea linear epitope or a conformational epitope.

As used herein, the terms “effective amount” and “effective dose” referto any amount or dose of a compound or composition that is sufficient tofulfill its intended purpose(s), i.e., a desired biological or medicinalresponse in a tissue or subject at an acceptable benefit/risk ratio. Forexample, in certain embodiments of the present invention, the intendedpurpose may be to inhibit activation of myostatin in vivo, to achieveclinically meaningful outcome associated with the myostatin inhibition.

Measure of the relevant intended purpose may be objective (i.e.,measurable by some assay or marker) or subjective (i.e., subject givesan indication of or feels an effect). In some embodiments, atherapeutically effective amount is an amount that, when administered toa patient population that meets certain clinical criteria for a disease,disorder or condition (for example, as determined by symptomsmanifested, disease progression/stage, genetic profile, etc.), astatistically significant therapeutic response is obtained among thepopulation.

In some embodiments, an effective amount is an amount that, whenadministered according to a particular regimen, produces a positiveclinical outcome with a reasonably acceptable level of adverse effects(e.g., toxicity), such that the adverse effects, if present, aretolerable enough for a patient to continue with the therapeutic regimen,and the benefit of the therapy overweighs risk of toxicity. Those ofordinary skill in the art will appreciate that in some embodiments ofthe invention, a unit dosage may be considered to contain an effectiveamount if it contains an amount appropriate for administration in thecontext of a dosage regimen correlated with a positive outcomc.

A therapeutically effective amount is commonly administered in a dosingregimen that may comprise multiple unit doses. For any particularpharmaceutical agent, a therapeutically effective amount (and/or anappropriate unit dose within an effective dosing regimen) may vary, forexample, depending on route of administration, on combination with otherpharmaceutical agents. In some embodiments, the specific therapeuticallyeffective amount (and/or unit dose) for any particular patient maydepend upon a variety of factors including the disorder being treatedand the severity of the disorder; the activity of the specificpharmaceutical agent employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and/or rate of excretion ormetabolism of the specific pharmaceutical agent employed; the durationof the treatment; and like factors as is well known in the medical arts.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences and fragments thereof. The humanantibodies of the disclosure may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo), for example in the CDRs and in particular CDR3. However, theterm “human antibody”, as used herein, is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “increase” in the context, e.g., of a disease symptom, such asfor example, a loss of function or loss of mass, e.g., muscle massassociated with a disease, refers to a statistically significantincrease in such level. The increase can be, for example, at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95%, or above the level of detection for the detectionmethod. The increase can also be, for example, about 1-10%, 10-20%,1-30%, 20-50%, 30-60%, 40-70%, 50-80%, or 60-90% above the level ofdetection for the detection method. In certain embodiments, the increaseis up to a level accepted as within the range of normal for anindividual without such disorder which can also be referred to as anormalization of a level. In certain embodiments, the increase is thenormalization of the level of a sign or symptom of a disease, anincrease in the difference between the subject level of a sign of thedisease and the normal level of the sign for the disease. In certainembodiments, the methods include an increase in the mass and/or functionof the muscle tissue after treatment of a subject with an antibody thatspecifically binds pro/latent myostatin. In certain embodiments, themethods include an increase in a level of pro-myostatin in a targetmuscle, as compared to a control level of pro-myostatin.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds pro/latent-myostatin is substantially free of antibodies thatspecifically bind antigens other than pro/latent-myostatin). An isolatedantibody that specifically binds pro/latent-myostatin may, however, havecross-reactivity to other antigens, such as pro/latent-myostatinmolecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

Unless explicitly stated otherwise, the term “mature myostatin” refersto a fully processed, biologically active form of myostatin, unlessexplicitly stated otherwise. A biologically active form of myostatin iscapable of myostatin receptor binding and/or activation. Wild typesequence of mature myostatin is provided as SEQ ID NO: 52. In somecases, mature myostatin may contain one or more mutations, which mayexhibit altered structure/function or stability.

As used herein, the term “myostatin inhibitor” refers to any compoundthat inhibits or antagonizes the activity or expression level ofmyostatin, e.g., pro/latent myostatin. In some embodiments, themyostatin inhibitor may be an antibody (including fragments thereof,such as Domain Antibodies (dAbs) as described in, for example, U.S. Pat.Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245), a smallmolecule inhibitor, an Adnectin, an Affibody, a DARPin, an Anticalin, anAvimer, a Versabody or a gene therapy. The antibody, or antigen bindingfragment thereof, may bind mature myostatin, a myostatin receptor,and/or GDF11. In some embodiments, the myostatin inhibitor is a smallmolecule inhibitor. In other embodiments, the myostatin inhibitor refersto a gene therapy. In one embodiment, the myostatin inhibitor bindsspecifically to myostatin, but not GDF11. In one embodiment, themyostatin inhibitor can be used to treat a metabolic disease, a musclecondition or disorder, a disease or disorder associated with an impairedneurological signaling or partial denervation or other conditiondescribed herein. In another embodiment, the myostatin inhibitor can beused to treat a disease involving fast twitch fibers, as describedherein. In another embodiment, a myostatin inhibitor can be used toprovide therapeutic effects below a lesion, as described herein.

As used herein, the phrase “latent myostatin in the circulation” or“circulating latent myostatin” refers to latent myostatin in the blood,plasma, or serum.

As used herein, the term “pro/latent-myostatin” refers to pro-myostatin,latent myostatin, or both (i.e., pro-forms or precursors of myostatin).

“Specific” and “specificity” in the context of an interaction betweenmembers of a specific binding pair (e.g., a ligand and a binding site,an antibody and an antigen, biotin and avidin) refer to the selectivereactivity of the interaction. The phrase “specifically binds to” andanalogous phrases refer to the ability of antibodies (or antigenicallyreactive fragments thereof) to bind specifically to an antigen (or afragment thereof) and not bind specifically to other entities. Specificbinding is understood as a preference for binding a certain antigen,epitope, receptor ligand, or binding partner with, for example, at least2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or1,000-fold preference over a control non-specific antigen, epitope,receptor ligand, or binding partner. “Specific binding” as used hereincan also refer to binding pairs based on binding kinetics such asK_(on), K_(off), and K_(D). For example, a ligand can be understood tobind specifically to its target site if it has a K_(off) of 10⁻² sec⁻¹or less, 10⁻³ sec⁻¹ or less, 10⁻⁴ sec⁻¹ or less, 10⁻⁵ sec⁻¹ or less, or10⁻⁶ sec⁻¹ or less; and/or a K_(D) of 10⁻⁶ M or less, 10⁻⁷M or less,10⁻⁸ M or less, 10⁻⁹M or less, 10⁻¹⁰ M or less, or 10⁻¹¹ M or less, or10⁻¹² M or less. It is understood that various proteins can share commonepitopes or other binding sites (e.g., kinase reactive sites). Incertain embodiments, binding sites may bind more than one ligand, butstill can be considered to have specificity based on binding preferenceas compared to a non-specific antigen and/or by having certain bindingkinetic parameters. Methods of selecting appropriate non-specificcontrols are within the ability of those of skill in the art. Bindingassays are typically performed under physiological conditions.

As used herein, the term “slow-twitch”, “slow twitch” “Type 1” or “TypeI” muscle refers to a muscle enriched in Type I muscle fibers and isused frequently, more postural, and help enable long-endurance featssuch as distance running. As used herein, the term “fast-twitch”, “fasttwitch” “Type 2” or “Type II” muscle refers to a muscle that provideshigher energy output and strength and is used in powerful bursts ofmovements like sprinting, but such a muscle fatigue faster and cannot beused repeatedly. Fast-twitch muscles break down into two categories offiber types: moderate fast-twitch fibers (Type IIA) and fast-twitchfibers (Type IIB or IIx). Moderate fast-twitch fibers are thicker,quicker to contract, and wear out more rapidly than slow-twitch fibers.Fast-twitch fibers, the most powerful and lowest in endurance, areactivated when the body nears maximum exertion. While most muscles tendto be comprised of a mixture of various fiber types, different musclescontain different ratios of fiber types. During development or inresponse to certain events (e.g., exercise, disease, injury, etc.),fiber types within a muscle or muscle group may undergo fiber typeswitching, resulting in an altered phenotype in muscle physiology.

As used herein, the term “subject” and “patient” may be usedinterchangeably. In one embodiment, a subject refers to a vertebrate, inparticular a mammal, in need of treatment, e.g., companion animals(e.g., dogs, cats and the like), farm animals (e.g., cows, pigs, horses,sheep, goats, poultry and the like) and laboratory animals (e.g., rats,mice, guinea pigs and the like). In some embodiments, the subject is ahuman who will benefit from or in need of treatment. In one embodiment,a subject is a human subject. In one embodiment, the subject is apediatric subject.

As used herein, the phrase “sustained increase” in the context ofincrease of muscle mass refers to an increase of muscle mass for aspecified time after the administration a therapeutically effectiveamount of a myostatin inhibitor, e.g., an anti-pro/latent-myostatinantibody, as described herein. Sustained increase may be continuous ornon-continuous, but overall results in an increase in muscle mass forthe specified time.

By “treating” or “preventing” a disease or disorder is meant delaying orpreventing the onset of such a disease or disorder, reversing,alleviating, ameliorating, inhibiting, slowing down or stopping theprogression, aggravation or deterioration, the progression or severityof a condition associated with such a disease or disordcr, but notnecessarily require a complete treatment or prevention of the disease ordisorder. In one embodiment, the symptoms of a disease or disorder arealleviated by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, or at least 50%.

Myostatin

Myostatin, also known as GDF8, is a member of the TGFβ superfamily, andbelongs to a subfamily including two members: myostatin and GDF11. Likeother members of the TGFβ superfamily, myostatin and GDF11 are bothinitially expressed as inactive precursor polypeptides (termedpro-myostatin and proGDF11, respectively). The domain structure andnomenclature are shown in FIG. 1A. FIG. 1B illustrates a cartoon modelof the overall structure of pro-myostatin, where the mature growthfactor is held locked in a cage comprised of two alpha helices connectedby a loop termed the “latency lasso”.

Myostatin is a well-characterized negative regulator of skeletal musclemass that is released from an autoinhibitory N-terminal prodomain by twoseparate protease cleavage steps. These cleavage events, within themuscle fiber microenvironment, for example, may be referred to assupracellular activation. Following activation, mature myostatin signalsby binding to a complex of Type I and II cell surface receptors (Alk4/5and ActRIIB) whose downstream signaling induces muscle atrophy. There isinterest in myostatin as a target for the treatment of muscle wasting. Anumber of therapeutics targeting the ActRIIB signaling pathway arecompleting early- to mid-stage clinical trials in muscle wastingconditions including sarcopenia, muscular dystrophies, cachexia, and hipreplacement/hip fracture. To date, the primary clinical strategy hasfocused on blocking the interaction between mature myostatin and cellsurface receptors. However, several therapeutic programs have beendiscontinued due to lack of specificity (leading to unacceptabletoxicities) and/or efficacy. In vivo, myostatin is primarily in complexwith its inhibitory prodomain.

Aspects of the disclosure provided herein relate to an assessment of theextent to which blocking the supracellular activation of myostatin fromthese inhibitory prodomain complexes provides a means for specificallyblocking myostatin pathway signaling. Further aspects of the disclosurerelate to an evaluation of a panel of human monoclonal antibodies thatselectively bind the myostatin precursor forms, including a subset thatinhibit proteolytic activation in vitro. In some embodiments, it hasbeen found that antibodies that block activation are capable ofprotecting mice from dexamethasone-induced muscle atrophy. Assessment ofserum and muscle samples from healthy animals and from those undergoingdexamethasone-induced atrophy demonstrated altered biodistribution ofprecursor forms during atrophy, a unique finding with importantimplications in understanding muscle wasting pathologies. Furthermore,treatment of healthy mice with a murine version of a potentactivation-blocking antibody promoted robust muscle growth and resultedin significant gains in muscle function. Results provided herein provideinsights into the significance of myostatin processing in skeletalmuscle protein homeostasis. In addition, blocking the supracellularactivation of the growth factor from precursor forms is a potent methodfor preventing myostatin signaling, a technique offering a noveltherapeutic strategy that can also be applied to other members of theTGFβ superfamily

Activation and release of mature myostatin is accomplished by severaldiscrete protease cleavage events. The first cleavage step ofpro-myostatin and proGDF11 is carried out by a proprotein convertase,which cuts at a conserved RXXR site between the prodomain and maturegrowth factor. This cleavage produces a “latent-myostatin,” in which themature myostatin is shielded from binding to its receptors by theprodomain. Activation and release of the mature, active myostatin growthfactor is accomplished after cleavage of latent-myostatin by anadditional protease from the BMP/tolloid family, such as mTLL-2. As usedherein, the term “mature myostatin” can refer to both full-length maturemyostatin, as well as fragments of the full-length mature myostatinwhich retain biological activity.

The term “pro-myostatin,” also known as “proGDF8,” refers to an inactiveprecursor of mature myostatin which comprises a disulfide-linkedhomodimer, each molecule of the homodimer comprising the amino terminalprodomain covalently bound to the carboxyl terminal mature myostatindomain. In one embodiment, “pro-myostatin” has not been cleaved byeither a proprotein convertase, or a protease from the BMP/tolloidfamily. Exemplary pro-myostatin sequences, variants thereof, and methodsof generating pro-myostatin are well known in the art and described inmore detail herein.

As used herein the term “latent-myostatin” refers to an inactiveprecursor of mature myostatin which comprises a disulfide-linkedhomodimer, each molecule of the homodimer comprising the amino terminalprodomain non-covalently bound to the carboxyl terminal mature myostatindomain. In one embodiment, “latent-myostatin” is generated from apro-myostatin that has been cleaved by a proprotein convertase, butwhich has not been cleaved by a protease from the BMP/tolloid family. Inanother embodiment, “latent-myostatin” can be generated by combining theprodomain and the carboxy terminal mature myostatin domain in vitro andallowing them to fold properly. See, for example, Sengle et al., J.Biol. Chem., 286(7):5087-5099, 2011. Exemplary latent-myostatinsequences, variants thereof, and methods of generating latent-myostatinare well known in the art and described in more detail herein.

Exemplary proGDF8 sequences in the human, rat, mouse and cynomolgus areprovided below. In these proGDF8 sequences, a proprotein convertasecleavage site is indicated in bold and a tolloid protease site isindicated by underlining. In some embodiments, the proprotein convertasecleavage site comprises amino acid residues 240 to 243 of SEQ ID NOs:52-55. In some embodiments, the tolloid protease site comprises aminoacid residues 74-75 of SEQ ID NOs: 52-55. It should be appreciated thatthe exemplary proGDF8 sequences provided herein are not intended to belimiting and additional proGDF8 sequences from other species, includingany isoforms thereof, are within the scope of this disclosure.

proGDF8 (human): (SEQ ID NO: 52)NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNISKDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS. proCDF8 (rat):(SEQ ID NO: 53) NEDSEREANVEKEGLCNACAWRQNTRYSRIEAIKIQILSKLRLETAPNISKDAIRQLLPRAPPLRELIDQYDVQRDDSSDGSLEDDDYAHTTETIITMPTESDFLMQADGKPKCCFFKFSSKIQYNKVVKAQLWIYLRAVKTPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMSPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCG CS. proGDF8 (mouse):(SEQ ID NO: 54) NEGSEREENVEKEGLCNACAWRQNTRYSRIEAIKIQILSKLRLETAPNISKDAIRQLLPRAPPLRELIDQYDVQRDDSSDGSLEDDDYAHTTETIITMPTESDFLMQADGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMSPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVGRCG CS.proGDF8 (cynomolgus): (SEQ ID NO: 55)NENSEQKENVEKEGLCNACTWRQNTKSSRIEAIKIQILSKLRLETAPNISKDAIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYAHTTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIA.

The prodomain of the myostatin polypeptide is compriscd of severalstructural domains as described previously (WO 2014/182676). Theseinclude, for example, Straight Jacket region, Fastner region, Armregion, Fingers region 1, Fingers region 2, Latency Loop, Alpha-1Helical region, and Bowtie region. In some embodiments, preferredantibodies or fragments thereof binds an epitope within the Arm regionof the myostatin prodomain. In some embodiments, the epitope includes atleast one amino acid residue from the “KALDEN” (SEQ ID NO: 118)polypeptide stretch within the Arm region of the prodomain. In someembodiments, the amino acid residue within the Arm region of theprodomain making contact with the antibody when bound to the antigen isa residue that is not conserved between myostatin and GDF11. In someembodiments, such residue(s) is/are K, E, and/or N of the polypeptidestretch (shown in bold type above).

Myostatin and GDF11 share a relatively high degree of conservationbetween their mature growth factor domains, with ninety percentidentity, but are much less well conserved in their prodomain regionswith less than fifty percent amino acid identity between the two.Myostatin and GDF11 bind and signal through the same receptorsconsisting of a Type I receptor (ALK4/5) in association with a type IIreceptor (ACTRIIA/B). Engagement of myostatin with Type I and Type IIreceptors initiates a signaling cascade leading to SMAD phosphorylationand transcriptional activation of muscle atrophy genes. The relativelyhigh degree of conservation in the mature growth factors has made itchallenging to identify reagents, such as monoclonal antibodies, thatcan differentiate between mature myostatin and GDF11.

In some embodiments, pro/latent-myostatin antibodies are provided hereinthat specifically bind to a chimeric construct that contains the growthfactor domain and N terminal propeptide portion of GDF11 and the Cterminal portion of the propeptide of GDF8. This chimeric construct, asforth below, is referred as GDF11 Arm8.

>GDF1 1 Arm8 (SEQ ID NO: 65)MDMRVPAQLLGLLLLWFSGVLGDYKDDDDKHHHHHHLEVLFQGPAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLKMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRRNLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQ QIIYGKIPGMVVDRCGCS

Role of Myostatin in Muscle Homeostasis and Metabolic Regulation

Skeletal muscle accounts for approximately 40% of body mass and is adynamic organ, turning over at a rate of 1-2% per day. Myostatin isbelieved to play a pivotal role in maintaining the homeostasis of muscleboth in healthy and disease conditions. Myostatin is capable of inducingmuscle atrophy via its inhibition of myoblast proliferation, increasingubiquitin-protcasomal activity and downregulating activity of theIGF-Akt pathway. These well-recognized effects are seen in multipleatrophy causing situations, including injury, diseases such as cachexia,disuse and space flight, demonstrating the importance of the myostatinsignaling mechanism. Based on this central role, significant work hasbeen pursued to inhibit myostatin's actions in viva. Indeed,antagonizing myostatin signaling has shown to favor musclegrowth/increase.

In addition, muscle is known to be the major protein reservoir of thebody and therefore contributes to amino acid homeostasis/metabolism.Along with glucose (made and stored as glycogen primarily in the liverand the muscles) and lipids (stored in fat tissues), proteins in musclescan act as an energy source (i.e., broken down to generate energy).Impairment or imbalance in the utilization or mobilization of theseenergy sinks in the body may, at least in part, underlie various typesof metabolic dysregulation. It is therefore contemplated that myostatinmay play a direct role in the regulation of metabolism by coordinatingthe balance between breakdown vs. synthesis/storage of glucose, fatsand/or muscles in the body. Indeed, while myostatin has been primarilyconsidered as a key regulator of muscle growth/loss since its discoveryin 1997, findings presented in more detail herein suggest a broader roleof myostatin as a metabolic regulator.

Because muscle homeostasis is correlated with amino acid/proteinmetabolism, it is further contemplated that myostatin inhibition may inturn regulate nitrogen metabolism and nitrogen mobilization in the body.In muscle catabolism, a muscle tissue breaks down into its buildingblocks, amino acids, which may be considered as a major reservoir (andthus a source) of nitrogen. Nitrogen is an element of ammonia, which ishighly toxic to the body and is excreted in a form of urea in humans.When nitrogen metabolism is dysregulated, possible outcome includes animbalance in fluid retention, which may manifest as systemic or localedema (e.g., congestion; fluid overload). Pulmonary edema, as well asrenal congestion, for example, is frequently observed in patients withheart failure, associated with decreased cardiac output. Pulmonarycongestion is in fact the most frequent cause of hospitalization in thisclinical setting and correlates with poor prognosis.

Similarly, in pathologic conditions that involve impairedosmoregulation, the affected individual may be particularly sensitive tosalt intake, which may cause or exacerbate fluid overload.

Therefore, for subjects with fluid retention or volume-overload, such assubjects having impaired osmoregulation and subjects with heart failure,e.g., chronic heart failure, current guidelines suggest thatdecongestion should be attempted using diuretic therapy (see, e.g.,Regolisti et al., Nephrology @ Point of Cre 2016; 2(1):e73-e87).However, in many cases, diuretic treatment is ineffective, or thesubject is refractory to diuretic therapy. Myostatin inhibitionaccording to the present disclosure may provide such patients withclinical benefits. Specifically, the methods of the present inventionare suitable for increasing responsiveness of subjects who arerefractory to diuretic treatment, or poorly responsive to diuretictreatment.

For example, administration of a myostatin inhibitor reduces thediuretic dose needed and/or offers improved control of symptoms, such asCHF symptoms; improves cardiac function; and/or prevents pathologiccardiac remodeling or other worsening of cardiac function chronically.Myostatin inhibition using an inhibitor described herein also reducesthe risk of CHF exacerbations, such as episodes of acute pulmonaryedema.

For other volume-overload states, e.g., renal failure or liver disease,which require high-dose diuretics, a myostatin inhibitor disclosedherein reduces the diuretic dose needed; offer improved control ofsymptoms, such as peripheral edema or congestion within the bodyinternally (including pleural effusions, ascites, hepatic congestion, oreyes-volume overload within the eyes, which can lead to retinaldetachment); and/or reduce the risk of pulmonary edema.

For subjects at higher risk for developing acute pulmonary edema, suchas subjects receiving IV fluids, blood transfusions, or fluid shifts,the myostatin inhibitors disclosed herein may be administeredprophylactically. For example, a subject with congestive heart failurewho needs to receive a blood transfusion can be prophylacticallyadministered a myostatin inhibitor during the blood transfusion toprevent the onset of acute pulmonary edema during the transfusion.

For subjects having CHF and/or other volume-overload states who develophyponatremia, either due to the volume-overload, itself, or fromdiuretics used to treat the volume-overload, myostatin inhibitorsdisclosed herein can be administered to treat the hyponatremia and/orenable higher doses of diuretics to be used, when diuretic dosing islimited by hyponatremia as a side effect. Generally speaking, however,the myostatin inhibitors disclosed herein may be used to treathyponatremia, irrespective of the underlying etiology.

Myostatin Pathway Inhibition

There are several myostatin pathway inhibitors, such as small molecules,antibodies or antigen-binding portions thereof, and gene therapies, invarious stages of clinical development towards the treatment ofmuscle-related conditions. Such pathway antagonists target either themature growth factor or its type II receptor. Notably, most of theseantagonists are not myostatin-specific, such that they antagonize thesignaling of multiple TGFβ family members. For example, a number ofcurrent clinical candidates block additional growth factors such asActivin A, GDF11, and BMPs 9 and 10, which are regulators ofreproductive biology, wound healing, erythropoiesis and blood vesselformation, respectively. Aspects of this disclosure relate to arecognition that the lack of specificity observed in these myostatinantagonists described elsewhere may pose a greater risk to certainpatient populations because they block additional biological pathwayssuch as those listed above in addition to myostatin. This may thereforepotentially limit the population of patients who can safely undergotherapy due to unacceptable adverse-effects such as abnormal bleeding,wound healing, or reproductive problems caused by off-target antibodybinding (Campbell, et al. Muscle Nerve (2016); David, L., Blood 109,1953-1961 (2007)). For example, Activin A is involved in both woundhealing and reproductive biology, and binding to Activin A wouldtherefore limit use in patients who have recently undergone surgery orinjury, or in women of reproductive age. Such increased risk of adverseeffects or toxicity may be particularly concerning where i) a patientpopulation requires a long-term treatment (such as chronic conditions);and/or, ii) a patient population is or includes pediatric patients, whomay be susceptible to such adverse effects and/or toxicity. Accordingly,the present invention includes a novel approach to inhibiting myostatinsignaling in vivo with potentially greater safety profiles.

Accordingly, provided herein are myostatin inhibitors, such asantibodies, or antigen binding fragments thereof, capable of binding topro-myostatin and/or latent myostatin, thereby inhibiting myostatinactivation, and uses thereof for treating diseases and disordersassociated with myopathy. In some embodiments, given the prevalence ofthe latent complex in circulation, treatments are provided herein thatspecifically target more abundant and longer-lived myostatin precursorse.g., pro-myostatin and latent myostatin, rather than the mature growthfactor. Without wishing to be bound by any particular theory, myostatininhibitors, such as antibodies, or antigen binding fragments thereof,provided herein may prevent the proteolytic activation of pro-myostatinand/or latent myostatin into mature myostatin which is considered the“active” form of myostatin, capable of activating the myostatin pathway,e.g., by binding Type I (ALK4/5) and Type II (ACTRIIA/B) receptors.

As used herein, the term “pro/latent-myostatin” refers to pro-myostatin,latent myostatin, or both. In some embodiments, an anti-pro/latentmyostatin antibody, or antigen binding fragment thereof, bindsspecifically to pro-myostatin. In some embodiments, an anti-pro/latentmyostatin antibody, or antigen binding fragment thereof, bindsspecifically to latent myostatin. In some embodiments, ananti-pro/latent myostatin antibody, or antigen binding fragment thereof,binds specifically to both latent myostatin and pro-myostatin. Inpreferred embodiments, the anti-pro/latcnt myostatin antibody, orantigen binding fragment thereof, that binds specifically topro-myostatin and/or latent myostatin does not bind mature myostatin. Inpreferred embodiments, the anti-pro/latent myostatin antibody, orantigen binding fragment thereof, that binds specifically topro-myostatin and/or latent myostatin does not bind pro/latent GDF11 ormature GDF11.

Anti-Pro/Latent Myostatin Antibodies, or Antigen-Binding Fragmentsthereof, and Production Thereof

The present disclosure is based, at least in part, on the surprisingdiscovery that blocking the activation step of myostatin, rather thantargeting already active myostatin, may provide an advantageous mode ofselectively inhibiting myostatin signaling in vivo. Thus, the inventionmay have utility as a therapeutic in any condition where selectivereduction of myostatin signaling in vivo is beneficial. Morespecifically, the invention includes surprising findings that specificinhibition of myostatin activation can effectuate not only muscle massincrease but also enhanced muscle function, as well as prevention ofmetabolic dysregulation. Unexpectedly, beneficial therapeutic effectscan also be achieved even below a lesion in a subject having impairedbut not complete loss of signaling between a neuron and a target tissue,such as a target muscle.

An antibody (interchangeably used in plural form) is an immunoglobulinmolecule capable of specific binding to a target, such as acarbohydrate, polynucleotide, lipid, polypeptide, etc., through at leastone antigen recognition site, located in the variable region of theimmunoglobulin molecule. An antibody includes an antibody of any class,such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and theantibody need not be of any particular class. Depending on the antibodyamino acid sequence of the constant domain of its heavy chains,immunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

Antibodies, or antigen binding fragments thereof, described herein arecapable of binding to a pro/latent-myostatin, thereby inhibiting theproteolytic activation of pro/latent-myostatin into mature myostatin. Insome instances, antibodies, or antigen binding fragments thereof,described herein can inhibit the proteolytic activation ofpro/latent-myostatin by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or higher. In some instances, antibodies described hereincan inhibit the proteolytic cleavage of pro-myostatin by a proproteinconvertase (e.g., furin) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or higher. In some instances, antibodies, or antigenbinding fragments thereof, described herein can inhibit the proteolyticcleavage of pro-myostatin or latent myostatin by a tolloid protease(e.g., mTLL2) by at least 20%, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, or higher.

In some embodiments, inhibition of proteolytic cleavage of pro-myostatinor latent myostatin by a tolloid protease results in a progressiveincrease in muscle mass. In some embodiments, a subject exhibits aprogressive increase in muscle mass for at least 2 weeks, 4 weeks, 6weeks, 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, or 20weeks (or any range bracketed by any of the values). The inhibitoryactivity of an anti-pro/latent-myostatin antibody can be measured byroutine methods, for example, by Western blot analysis as described inExample 1 and FIG. 3 disclosed in WO 2016/073853, the entire contents ofwhich are expressly incorporated herein by reference. However, it shouldbe appreciated that additional methods may be used for measuring theinhibitory activity of an anti-pro/latent-myostatin antibody onproteolytic cleavage of pro/latent-myostatin. In some embodiments,inhibition of pro/latent-myostatin cleavage (e.g., by a proproteinconvertase and/or tolloid protease) may be reflected as an inhibitionconstant (Ki), which provides a measure of inhibitor potency, and whichit is the concentration of inhibitor (e.g., an anti-pro/latent-myostatinantibody) required to reduce protease activity (e.g., of a proproteinconvertase or tolloid protease) by half and is not dependent on enzymeor substrate concentrations.

In some embodiments, a proprotein convertase comprises (i) a catalyticdomain that hydrolyzes a peptide bond of a protein containing aproprotein convertase cleavage site, and (ii) a binding pocket thatbinds to an rTGF with a proprotein convertase cleavage site. Examples ofproprotein convertases for use in accordance with the present disclosureinclude, without limitation, PCSK5/6, PACE4, PACE7 and PACE3 (e.g.,furin). A proprotein convertase, in some embodiments, is obtained, e.g.,purified from, any mammal including, without limitation, humans, monkeysor rodents (e.g., mice, rats, hamsters). In another embodiment, aproprotein convertase is produced recombinantly.

In some embodiments, a proprotein convertase is homologous to aproprotein convertase selected from the group consisting of: PCSK5/6,PACE4, PACE7 and PACE3 (e.g., furin). For example, a proproteinconvertase may be at least 70% identical, at least 80% identical, atleast 90% identical, at least 95% identical, at least 96% identical, atleast 97% identical, at least 98% identical, at least 99% identical, atleast 99.5% identical, or at least about 99.9% identical to PCSK5/6,PACE4, PACE7 or PACE3 (e.g., furin).

A proprotein convertase cleavage site, in some embodiments, is an aminosequence that can be cleaved by a proprotein convertase (e.g., PCSK5/6,PACE4, PACE7 and PACE3). In some embodiments, the proprotein convertasecleavage site comprises the amino acid sequence R-X-X-R, where R isarginine and X is any amino acid. In some embodiments, the proproteinconvertase cleavage site comprises the amino acid sequence R-X-(K/R)-R,where R is arginine, K is lysine and X is any amino acid. In someembodiments, the proprotein convertase cleavage site comprises the aminoacid sequence is R—V-R-R (SEQ ID NO: 57), where R is arginine and V isvaline Exemplary proprotein convertase cleavage sites for human, rat,mouse, and cynomolgus myostatin are shown, in bold, in SEQ ID NOs:52-55. In some embodiments, the proprotein convertase cleavage sitecomprises the amino acid sequence RSRR (SEQ ID NO: 56).

In some embodiments, tolloid proteases for use in accordance with thepresent disclosure include, without limitation, BMP-1, mTLL-1 andmTLL-2. A tolloid protease may be obtained from any mammal including,without limitation, humans, monkeys, or rodents (e.g., mice, rats,hamsters).

In some embodiments, a tolloid protease is homologous to a tolloidprotease selected from the group consisting of: BMP-1, mTLL-1 andmTLL-2. For example, a tolloid protease may be at least 70% identical,at least 80% identical, at least 90% identical, at least 95% identical,at least 96% identical, at least 97% identical, at least 98% identical,at least 99% identical, at least 99.5% identical, or at least about99.9% identical to BMP-1, mTLL-1 and mTLL-2.

A tolloid protease cleavage site, in some embodiments, is an aminosequence that can be cleaved by a tolloid (e.g., BMP-1, mTLL-1 andmTLL-2). Exemplary tolloid protease cleavage sites for human, rat,mouse, and cynomolgus myostatin are shown, in underlining, in SEQ IDNOs: 52-55. In some embodiments, the tolloid cleavage site comprises theamino acid sequence QR, where Q is glutamine and R is arginine.

In some embodiments, antibodies, or antigen binding fragments thereof,described herein are capable of binding to a pro/latent-myostatin,thereby inhibiting myostatin activity. In some instances, theantibodies, or antigen binding fragments thereof, described herein caninhibit myostatin signaling by at least 20%, e.g., 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, or higher. In some embodiments, inhibition ofMyostatin signaling can be measured by routine methods, for example,using a myostatin activation assay as described in Example 1 disclosedin WO 2016/073853, the entire contents of which are expresslyincorporated herein by reference. However, it should be appreciated thatadditional methods may be used for measuring myostatin signalingactivity.

It should be appreciated that the extent of proteolytic cleavage ofmyostatin, e.g., by a proprotein convertase and/or a tolloid protease,can be measured and/or quantified using any suitable method. In someembodiments, the extent of proteolytic cleavage of myostatin is measuredand/or quantified using an enzyme-linked immunosorbent assay (ELISA).For example, an ELISA may be used to measure the level of releasedgrowth factor (e.g., mature myostatin). As another example, an antibody,or antigen binding fragment thereof, that specifically binds topro-myostatin, latent myostatin and/or mature myostatin can be used inan ELISA to measure the level of a specific form of myostatin (e.g.,pro/latent/mature-myostatin), to quantify the extent of proteolyticcleavage of myostatin. In some embodiments, the extent of proteolyticcleavage of myostatin is measured and/or quantified usingimmunoprecipitation followed by SDS-PAGE or mass spectrometry of trypticpeptides, fluorescence anisotropy-based techniques, FRET assays,hydrogen-deuterium-exchange mass spectrometry, and/or NMR spectroscopy.

In some embodiments, antibodies, also known as immunoglobulins, aretetrameric glycosylated proteins composed of two light (L) chains ofapproximately 25 kDa each and two heavy (H) chains of approximately 50kDa each. Two types of light chain, termed lambda and kappa, may befound in antibodies. Depending on the amino acid sequence of theconstant domain of heavy chains, immunoglobulins can be assigned to fivemajor classes: A, D, E, G, and M, and several of these may be furtherdivided into subclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁,and IgA₂. Each light chain typically includes an N-terminal variable (V)domain (V_(L)) and a constant (C) domain (C_(L)). Each heavy chaintypically includes an N-terminal V domain (V_(H)), three or four Cdomains (C_(H)1-3), and a hinge region. The C_(H) domain most proximalto V_(H) is designated as C_(H)1. The V_(H) and V_(L) domains consist offour regions of relatively conserved sequences called framework regions(FR1, FR2, FR3, and FR4), which form a scaffold for three regions ofhypervariable sequences (complementarity determining regions, CDRs). TheCDRs contain most of the residues responsible for specific interactionsof the antibody with the antigen. CDRs are referred to as CDR1, CDR2,and CDR3. Accordingly, CDR constituents on the heavy chain are referredto as CDRH1, CDRH2, and CDRH3, while CDR constituents on the light chainare referred to as CDRL1, CDRL2, and CDRL3. The CDRs typically refer tothe Kabat CDRs, as described in Sequences of Proteins of ImmunologicalInterest, US Department of Health and Human Services (1991), eds. Kabatet al. Another standard for characterizing the antigen binding site isto refer to the hypervariable loops as dcscribcd by Chothia. See, e.g.,Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson etal. (1995) EMBO J. 14:4628-4638. Still another standard is the AbMdefinition used by Oxford Molecular's AbM antibody modeling software.See, generally, e.g., Protein Sequence and Structure Analysis ofAntibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.:Duebel, S, and Kontcrmann, R., Springer-Verlag, Heidelberg). Embodimentsdescribed with respect to Kabat CDRs can alternatively be implementedusing similar described relationships with respect to Chothiahypervariable loops or to the AbM-defined loops, or combinations of anyof these methods.

Anti-pro/latent-myostatin antibodies, or antigen binding fragmentsthereof, suitable for use in the methods of the present inventioninclude those described in International Patent Application Nos.PCT/US15/59468 and PCT/US16/52014. The entire contents of each of theforegoing applications are incorporated herein by reference in theirentireties.

In some embodiments, anti-pro/latent-myostatin antibodies, or antigenbinding fragments thereof, of the present disclosure and the nucleicacid molecules of the present disclosure that encode the antibodies, orantigen binding fragments thereof, include the CDR amino acid sequencesshown in Tables 1-3.

TABLE 1 CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID Antibody NOs: 1-3) NOs: 4-9) NOs: 10-11)NOs: 12-17) NOs: 18-21) NOs: 22-23) Ab1 SSYGMH VISYDGSNK DLLVRFLEWSHSGSSSNIGSNT SDNQRPS AAWDDSLNGV Kabat: (SEQ ID YYADSVKG YYGMDV VH (SEQ ID(SEQ ID IMGT: NO: 1) (SEQ ID (SEQ ID (SEQ ID NO: 18) NO: 22) GFTFSSYGMNO: 4) NO: 10) NO: 12) SDN H ISYDGSN SSNIGSNT (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 19) NO: 2) NO: 5) NO: 13) Ab2 SSYGMH VISYDGSNK DLLVRFLEWSHSGSSSNIGSNT SDNQRPS AAWDDSLNGV Kabat: (SEQ ID YYADSVKG YYGMDV VH (SEQ ID(SEQ ID IMGT: NO: 1) (SEQ ID (SEQ ID (SEQ ID NO: 18) NO: 22) GFTFSSYGMNO: 4) NO: 10) NO: 12) SDN H ISYDGSN SSNIGSNT (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 19) NO: 2) NO: 5) NO: 13) Ab3 SSYGMH VISYDGSIK DLLVRFLEWSHSGSTSNIGSVT SDDQRPS AAWDESLNGV Kabat: (SEQ ID YYADSVKG KYGMDV VH (SEQ ID(SEQ ID IMGT: NO: 1) (SEQ ID (SEQ ID (SEQ ID NO: 20) NO: 23) GFAFSSYGMNO: 6) NO: 11) NO: 14) SDD H ISYDGSI TSNIGSNT (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 21) NO: 3) NO: 7) NO: 15) Ab4 SSYGMH VISYDGSIK DLLVRFLEWSHSGSTSNIGSVT SDDQRPS AAWDESLNGV Kabat: (SEQ ID YYADSVKG KYGMDV VH (SEQ ID(SEQ ID IMGT: NO: 1) (SEQ ID (SEQ ID (SEQ ID NO: 20) NO: 23) GFAFSSYGMNO: 6) NO: 11) NO: 14) SDD H ISYDGSI TSNIGSNT (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 21) NO: 3) NO: 7) NO: 15) Ab5 SSYGMH VISYDGNNK DLLVRFLEWSHSGSSSNIGGVT SDDQRPS AAWDESLNGV Kabat: (SEQ ID YYADSVKG KYGMDV VH (SEQ ID(SEQ ID IMGT: NO: 1) (SEQ ID (SEQ ID (SEQ ID NO: 20) NO: 23) GFAFSSYGMNO: 8) NO: 11) NO: 16) SDD H ISYDGNN SSNIGGNT (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 21) NO: 3) NO: 9) NO: 17)

In Table 1, the single sequences of CDRH3 and CDRL3 reflect Kabat andIMGT.

TABLE 2 Amino Acid Sequence Nucleic Acid Sequence Description(SEQ ID NO) (SEQ ID NO) Heavy chain QIQLVQSGGGVVQPGRSLRCAGATCCAGCTGGTGCAGTCTGGGGGAGGCG variable region LSCAASGFTFSSYGMHWVRTGGTCCAGCCTGGGAGGTCCCTGAGACTCTC - Ab1 parental QAPGKGLEWVAVISYDGSNCTGTGCAGCGTCTGGATTCACCTTCAGTAGC KYYADSVKGRFTISRDNSKTATGGCATGCACTGGGTCCGCCAGGCTCCAG NTLYLQMNSLRAEDTAVYYGCAAGGGGCTGGAGTGGGTGGCAGTTATATC CARDLLVRFLEWSHYYGMDATATGATGGAAGTAATAAATACTATGCAGAC VWGQGTTVTVSSTCCGTGAAGGGCCGATTCACCATCTCCAGAG (SEQ ID NO: 24)ACAATTCCAAGAACACGCTGTATCTGCAAAT GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGAT TTTTGGAGTGGTCGCACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCA (SEQ ID NO: 38) Heavy chainQVQLVESGGGVVQPGRSLR CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCG variable regionLSCAASGFTFSSYGMHWVR TGGTCCAGCCTGGGAGGTCCCTGAGACTCTC - Ab2QAPGKGLEWVAVISYDGSN CTGTGCAGCGTCTGGATTCACCTTCAGTAGC germlineKYYADSVKGRFTISRDNSK TATGGCATGCACTGGGTCCGCCAGGCTCCAG NTLYLQMNSLRAEDTAVYYGCAAGGGGCTGGAGTGGGTGGCAGTTATATC CARDLLVRFLEWSHYYGMDATATGATGGAAGTAATAAATACTATGCAGAC VWGQGTTVTVSSTCCGTGAAGGGCCGATTCACCATCTCCAGAG (SEQ ID NO: 25)ACAATTCCAAGAACACGCTGTATCTGCAAAT GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGAT TTTTGGAGTGGTCGCACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCA (SEQ ID NO: 39) Heavy chainQIQLVQSGGGVVQPGRSLR CAGATCCAGCTGGTGCAGTCTGGGGGAGGCG variable regionLSCAASGFAFSSYGMHWVR TGGTCCAGCCTGGGAGGTCCCTGAGACTCTC - Ab3 parentalQAPGKGLEWVAVISYDGSI CTGTGCAGCGTCTGGATTCGCCTTCAGTAGC KYYADSVKGRFTISRDNSKTATGGCATGCACTGGGTCCGCCAGGCTCCAG NTLYLQMNSLRAEDTAVYYGCAAGGGGCTGGAGTGGGTGGCAGTTATATC CARDLLVRFLEWSHKYGMDATATGATGGAAGTATCAAATACTATGCAGAC VWGQGTTVTVSSTCCGTGAAGGGCCGATTCACCATCTCCAGAG (SEQ ID NO: 26)ACAATTCCAAGAACACGCTGTATCTGCAAAT GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGAT TTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCA (SEQ ID NO: 40) Heavy chainQVQLVESGGGVVQPGRSLR CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCG variable regionLSCAASGFAFSSYGMHWVR TGGTCCAGCCTGGGAGGTCCCTGAGACTCTC - Ab4QAPGKGLEWVAVISYDGSI CTGTGCAGCGTCTGGATTCGCCTTCAGTAGC germlineKYYADSVKGRFTISRDNSK TATGGCATGCACTGGGTCCGCCAGGCTCCAG NTLYLQMNSLRAEDTAVYYGCAAGGGGCTGGAGTGGGTGGCAGTTATATC CARDLLVRFLEWSHKYGMDATATGATGGAAGTATCAAATACTATGCAGAC VWGQGTTVTVSSTCCGTGAAGGGCCGATTCACCATCTCCAGAG (SEQ ID NO: 27)ACAATTCCAAGAACACGCTGTATCTGCAAAT GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGAT TTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCA (SEQ ID NO: 41) Heavy chainQIQLVQSGGGVVQPGRSLR CAGATCCAGCTGGTGCAGTCTGGGGGAGGCG variable regionLSCAASGFAFSSYGMHWVR TGGTCCAGCCTGGGAGGTCCCTGAGACTCTC - Ab5 parentalQAPGKGLEWVAVISYDGNN CTGTGCAGCGTCTGGATTCGCCTTCAGTAGC KYYADSVKGRFTISRDNSKTATGGCATGCACTGGGTCCGCCAGGCTCCAG NTLYLQMNSLRAEDTAVYYGCAAGGGGCTGGAGTGGGTGGCAGTTATATC CARDLLVRFLEWSHKYGMDATATGATGGAAATAATAAATACTATGCAGAC VWGQGTTVTVSSTCCGTGAAGGGCCGATTCACCATCTCCAGAG (SEQ ID NO: 28)ACAATTCCAAGAACACGCTGTATCTGCAAAT GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGAT TTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCA (SEQ ID NO: 42) Heavy chainQVQLVESGGGVVQPGRSLR CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCG variable regionLSCAASGFAFSSYGMHWVR TGGTCCAGCCTGGGAGGTCCCTGAGACTCTC - Ab6QAPGKGLEWVAVISYDGNN CTGTGCAGCGTCTGGATTCGCCTTCAGTAGC germlineKYYADSVKGRFTISRDNSK TATGGCATGCACTGGGTCCGCCAGGCTCCAG NTLYLQMNSLRAEDTAVYYGCAAGGGGCTGGAGTGGGTGGCAGTTATATC CARDLLVRFLEWSHKYGMDATATGATGGAAATAATAAATACTATGCAGAC VWGQGTTVTVSSTCCGTGAAGGGCCGATTCACCATCTCCAGAG (SEQ ID NO: 29)ACAATTCCAAGAACACGCTGTATCTGCAAAT GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCTCCTGGTGCGAT TTTTGGAGTGGTCGCACAAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCA (SEQ ID NO: 43) Light chainQPVLTQPPSASGTPGQRVT CAGCCTGTGCTGACTCAGCCACCCTCAGCGT variable regionISCSGSSSNIGSNTVHWYQ CTGGGACCCCCGGGCAGAGGGTCACCATCTC - Ab1 parentalQLPGTAPKLLIYSDNQRPS TTGTTCTGGAAGCAGCTCCAACATCGGAAGT GVPDRFSGSKSGTSASLVIAATACTGTCCACTGGTACCAGCAACTCCCAG SGLQSDDEADYYCAAWDDSGAACGGCCCCCAAACTCCTCATCTATAGTGA LNGVFGGGTKLTVLTAATCAGCGCCCCTCAGGGGTCCCTGACCGA (SEQ ID NO: 30)TTCTCTGGCTCCAAGTCTGGCACCTCAGCCT CCCTGGTCATCAGTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGAT GACAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 44) Light chain QSVLTQPPSASGTPGQRVTCAGTCTGTGCTGACTCAGCCACCCTCAGCGT variable region ISCSGSSSNIGSNTVHWYQCTGGGACCCCCGGGCAGAGGGTCACCATCTC - Ab2 QLPGTAPKLLIYSDNQRPSTTGTTCTGGAAGCAGCTCCAACATCGGAAGT germline GVPDRFSGSKSGTSASLAIAATACTGTCCACTGGTACCAGCAACTCCCAG SGLQSEDEADYYCAAWDDSGAACGGCCCCCAAACTCCTCATCTATAGTGA LNGVFGGGTKLTVLTAATCAGCGCCCCTCAGGGGTCCCTGACCGA (SEQ ID NO: 31)TTCTCTGGCTCCAAGTCTGGCACCTCAGCCT CCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGAT GACAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 45) Light chain QPVLTQPPSASGTPGQRVTCAGCCTGTGCTGACTCAGCCACCCTCAGCGT variable region ISCSGSTSNIGSNTVHWYQCTGGGACCCCCGGGCAGAGGGTCACCATCTC - Ab3 parental QLPGTAPKLLIYSDDQRPSTTGTTCTGGAAGCACCTCCAACATCGGAAGT GVPDRFSGSKSGTSASLVIAATACTGTCCACTGGTACCAGCAACTCCCAG SGLQSDDEADYYCAAWDESGAACGGCCCCCAAACTCCTCATCTATAGTGA LNGVFGGGTKLTVLTGATCAGCGCCCCTCAGGGGTCCCTGACCGA (SEQ ID NO: 32)TTCTCTGGCTCCAAGTCTGGCACCTCAGCCT CCCTGGTCATCAGTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGAT GAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 46) Light chain QSVLTQPPSASGTPGQRVTCAGTCTGTGCTGACTCAGCCACCCTCAGCGT variable region ISCSGSTSNIGSNTVHWYQCTGGGACCCCCGGGCAGAGGGTCACCATCTC - Ab4 QLPGTAPKLLIYSDDQRPSTTGTTCTGGAAGCACCTCCAACATCGGAAGT germline GVPDRFSGSKSGTSASLAIAATACTGTCCACTGGTACCAGCAACTCCCAG SGLQSEDEADYYCAAWDESGAACGGCCCCCAAACTCCTCATCTATAGTGA LNGVFGGGTKLTVLTGATCAGCGCCCCTCAGGGGTCCCTGACCGA (SEQ ID NO: 33)TTCTCTGGCTCCAAGTCTGGCACCTCAGCCT CCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGAT GAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 47) Light chain QPVLTQPPSASGTPGQRVTCAGCCTGTGCTGACTCAGCCACCCTCAGCGT variable region ISCSGSSSNIGGNTVHWYQCTGGGACCCCCGGGCAGAGGGTCACCATCTC - Ab5 parental QLPGTAPKLLIYSDDQRPSTTGTTCTGGAAGCAGCTCCAACATCGGAGGA GVPDRFSGSKSGTSASLVIAATACTGTCCACTGGTACCAGCAACTCCCAG SGLQSDDEADYYCAAWDESGAACGGCCCCCAAACTCCTCATCTATAGTGA LNGVFGGGTKLTVLTGATCAGCGCCCCTCAGGGGTCCCTGACCGA (SEQ ID NO: 34)TTCTCTGGCTCCAAGTCTGGCACCTCAGCCT CCCTGGTCATCAGTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGTGCAGCATGGGAT GAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 48) Light chain QSVLTQPPSASGTPGQRVTCAGTCTGTGCTGACTCAGCCACCCTCAGCGT variable region ISCSGSSSNIGGNTVHWYQCTGGGACCCCCGGGCAGAGGGTCACCATCTC - Ab6 QLPGTAPKLLIYSDDQRPSTTGTTCTGGAAGCAGCTCCAACATCGGAGGA germline GVPDRFSGSKSGTSASLAIAATACTGTCCACTGGTACCAGCAACTCCCAG SGLQSEDEADYYCAAWDESGAACGGCCCCCAAACTCCTCATCTATAGTGA LNGVFGGGTKLTVLTGATCAGCGCCCCTCAGGGGTCCCTGACCGA (SEQ ID NO: 35)TTCTCTGGCTCCAAGTCTGGCACCTCAGCCT CCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGAT GAGAGCCTGAATGGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 49) Ab2-Heavy QVQLVESGGGVVQPGRSLR ChainLSCAASGFTFSSYGMHWVR QAPGKGLEWVAVISYDGSN KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY CARDLLVRFLEWSHYYGMD VWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKY GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVI CVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG (SEQ ID NO: 50) Ab2-LightQSVLTQPPSASGTPGQRVT Chain ISCSGSSSNIGSNTVHWYQ QLPGTAPKLLIYSDNQRPSGVPDRFSGSKSGTSASLAI SGLQSEDEADYYCAAWDDS LNGVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKA TLVCLISDFYPGAVTVAWK ADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKS HRSYSCQVTHEGSTVEKTV APTECS (SEQ ID NO: 51)

TABLE 3 SEQ ID NOs: from left to CDR-H3 CDR-L3 VH VL scFV right Ab7ESLIRF NSWTRS QVQLQQSGAEVKKPG QSALTQPASVSGSPGQSL QVQLQQSGAEVKKPGASVKVSC71-75 LEDPQQ NNYI ASVKVSCKASGYTFT TISCTGTSSDIGGYNYVSKASGYTFTSYYMHWVRQAPGQG GGMDV SYYMHWVRQAPGQGL WYQQHPGKAPKLIIYDVTLEWMGIINPSGGSTSYAQKFQG EWMGIINPSGGSTSY DRPSGVSGRFSGSKSGNTRVTMTRDTSTSTVYMELSSLRS AQKFQGRVTMTRDTS ASLTISGLQTEDEAEYFCEDTAVYYCARESLIRFLEDPQQ TSTVYMELSSLRSED NSWTRSNNYIFGGGTKLTGGMDVWGQGTTVTVSSGSASAP TAVYYCARESLIRFL VLGQPKAAPSVTLTLGGGGSGGGGSAAAQSALTQP EDPQQGGMDVWGQGT ASVSGSPGQSLTISCTGTSSDI TVTVSSGGYNYVSWYQQHPGKAPKLIIY DVTDRPSGVSGRFSGSKSGNTA SLTISGLQTEDEAEYFCNSWTRSNNYIFGGGTKLTVLGQPKAAP SVTLFPPSS Ab10 DRYSSS QSYDAS EVQLVQSGGGVVQSGNFMLTQPHSVSESPGRTV EVQLVQSGGGVVQSGRSLRLSC 76-80 WGGGFD SLWVRSLRLSCVASGFSFS TIPCSGRGGSIASDSVQW VASGFSFSNYGMHWVRQAPGKG YNYGMHWVRQAPGKGL YQQRPGSAPTTIIYEDNQ LEWLARIWYDGSNKWYADSVKGEWLAFIWYDGSNKWY RPSGVPDRFSGSVDSSSN RFTISRDNSKNALYLQMNSLRAADSVKGRFTISRDNS SASLTISGLRTEDEADYY EDTAVYYCARDRYSSSWGGGFDKNALYLQMNSLRAED CQSYDASSLWVFGGKTKL YWGQGTVLTVSSGSASAPTLGGTAVYYCARDRYSSSW TVLGQPKAAPSVTL GGSGGGGSAAANFMLTQPHSVS GGGFDYWGQGTVLTVESPGRTVTIPCSGRGGSIASDS SS VQWYQQRPGSAPTTIIYEDNQR PSGVPDRFSGSVDSSSNSASLTISGLRTEDEADYYCQSYDASSL WVFGGKTKLTVLGQPKAAPSVT LFPPSSKASGA Ab11 DRHSLGQAWDST QLQLQQSGGGLVKPG SSELTQPSVSVSPGQTAT QLQLQQSGGGLVKPGGLSLRLS 81-85DFDY TVV GSLRLSCAASGFTFS ITCSGDKLGDKYASWYQQ CAASGFTFSSYSMNWVRQAPGKSYSMNWVRQAPGKGL KPGQSPVLVIYQDTKRPS GLEWVSSISSSSSYIYYADSVKEVWSSISSSSSYIYY GIPARFSGSNSGNTATLT GRFTISRDNAKNSLYLQMNSLRADSVKGRFTISRDNA ISGTQAMDEAAYYCQAWD AEDTAVYYCVRDRHSLGDFDYWKNSLYLQMNSLRAED STTVVFGGGTKLTVLGQP GQGTLVTVSSGSASAPTLGGGGTAVYYCVRDRHSLGD KAAPSVTLFPPSS SGGGGSAAASSELTQPPSVSVS FDYWGQGTLVTVSSGPGQTATITCSGDKLGDKYASWY S QQKPGQSPVLVIYQDTKRPSGI PARF Ab9 HGLMDD ATWDDSQVQLVQSGAEVKKPG QPVLTQPPSASGTPGQRV QVQLVQSGAEVKKPGSSVKVSC 86-90 SSGYYLLTGVV SSVKVSCKASGGTFS TISCSGSSSNIGSNTVEW KASGGTFSSYAISWVRQAPGQG SNAFDISYAISWVRQAPGQGL YQQLPGTAPKLLIHSNNQ LEWMGGIIPIFTANYAQKFQGREWMGGIIPIFGTANY RPSGVPDRFSGSRSGTSA VTITADESTSTAYMELSSLRSEAQKFQGRVTITADES SLAISGLQSEDEADYFCA DTAVYYCANHGLMDDSSGYYLSTSTAYMELSSLRSED TWDDSLTGVVFGGGTTLT NAFDIWGQGTMVTVSSGSASAPTAVYYCANHGLMDDS VLGQPKAAPSVTLFPPSS TLGGGGSGGGGSAAAQPVLTQPSGYYLSNAFDIWGQG PSASGTPGQRVTISCSGSSSNI TMVTVSSGS GSNTVEWYQQLPGTAPKLLIHSNNQRPSGVPDRFSGSRSGTSAS LAISGLQSEDEADYFCATWDDS LTGVVFGGGTTLTVLGQPKAAPSVTLFPPSS Ab12 VGTAAA AAWDDS QVQLVQSGGGLIQPG QPVLTQPPSASGTPGQRVQVLQVQSGGGLIQPGGSLRLSC 91-95 GDAFDI LSGWV GSLRLSCAASGFTVSTISVFGSSSNIGSNYVYW AASGFTVSSYSMNWVRQAPGKG SYSMNWVRQAPGKGLYQQLPGTAPKLLIYRNNQ LEWVSYISSSGSTIYYADSVKG EWVSYISSSGSTIYYRPSGVPDRFSGSKSGTSA RFTISRDNAKNSLYLQMNSLRA ADSVKGRFTISRDNASLAISGLRSEDEADYYCA EDTALYYCAKVGTAAAGDAFDI KNSLYLQMNSLRAEDAWDDSLSGWVFGGGTKLT WGQGTMVTVSSGSASAPTLGGG TALYYCAKVGTAAAGVLGQPKAAPSVTLFPPSS GSGGGGSAAAQPVLTQPPSASG DAFDIWGQGTMVTVSTPGQRVTISCFGSSSNIGSNYV SGS YWYQQLPGTAPKLLIYRNNQRP SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWV FGGGTKLTVLGQPKAAPSVTLF PPSS Ab8 VGFYDY QQYGTSQIQLVQSGAEVKKPG EIVMTQSPGTLSLSPGER QIQLVQSGAEVKKPGASVKVSC  96-100 VWGSYPPLT ASVKVSCKASGYTFT ATLSCRASQSVSSNYLAW KASGYTFTSYGISWVRQAPGQG YDAFDISYGISWVRQAPGQGL YQQKPGQAPRLLIYDASN LEWMGWISAYNGNTNYAQKLQGEWMGWISAYNGNTNY RATGIPARFSGSGSGTDF RVTMTTDTSTSTAYMELSSLRSAQKLQGRVTMTTDTS TLTISSLEPEDFALYYCQ EDTAVYYCARVGFYDYVWGSYPTSTAYMELSSLRSED QYGTSPLTFGGGTKLEIK YDAFDIWGQGTMVTVSSGSASATAVYYCARVGFYDYV PTLGGGGSGGGGSAAAEIVMTQ WGSYPYDAFDIGSYPSPGTLSLSPGERATLSCRASQS YDAFDIWGQGTMVTV VSSNYLAWYQQKPGQAPRLLIY SSDASNRATGIPARFSGSGSGTDF TLTISSLEPEDFALYYCQQYGT SPLTFGGGTKLEIKRTVAAPSV FAb13 DTSNGG SSYTSS EVQLVQSGGGLVQPG QSALTQPASVSGSPGQSIEVQLVQSGGGLVQPGRSLRLSC 101-105 YSSSSF STLV RSLRLSCAASGFTFDTISCTGTSSDVGGYNYVS AASGFTFDDYAMHWVRQAPGKG DY DYAMHWVRQAPGKGLWYQQHPGTAPKLMIYDVS LEWVSGISWNSGSIGYADSVKG EWVSGISWNSGSIGYYRPSGVSNRFSGSKSGNT RFTISRDNAKNSLYLQMNSLRA ADSVKGRFTISRDNAASLTISGLQAEDEADYYC EDTALYYCAKDTSNGGYSSSSF KNSLYLQMNSLRAEDSSYTSSSTLVFGTGTKVT DYWGQGTLVTVSSGSASAPTLG TALYYCAKDTSNGGY VLGGGSGGGGSAAAQSALTQPASV SSSSFDYWGQGTLVT SGSPGQSITISCTGTSSDVGGY VSSNYVSWYQQHPGTAPKLMIYDVS YRPSGVSNRFSGSKSGNTASLT ISGLQAEDEADYYCSSYTSSSTLVFGTGTKVTVLGQPKANPTVT LFPPSS Ab14 LVYGGY AAWDDS EVQLLESRAEVKKPGEVQLLESRAEVKKPGESL EVQLLERSRAEVKKPGESLKIS 106-110 DEPGYY LNGWVESLKISCKGSGYSFT KISCKGSGYSFTSYWIGW CKGSGYSFTSYWIGWVRQMPGK FDYSYWIGWVRQMPGKGP VRQMPGKGPEWMGIIYPG GPEWMGIIYPGDSDTRYSPSFQEWMGIIYPGDSDTRY DSDTRYSPSFQGQVTISA GQVTISADKSISTAYLQWSSLKSPSFQGQVTISADKS DKSISTAYLQWSSLKASD ASDTAMYYCARLVYGGYDEPGYISTAYLQWSSLKASD TAMYYCARLVYGGYDEPG YFDYWGQGTLVTVSSGSASAPTTAMYYCARLVYGGYD YYFDYWGQGTLVTVSS LGGGGSGGGGSAAAQSVLTQPP EPGYYFDYWGQGTLVSASGTPGQRVTISCSGSSSNIR TVSS SNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSEDEADY YCAAWDDSLNGWVFGGGTKLTV LGQPKAAPSVTLFPPSSKASGAAb15 VDGLEY SSYAGS EVQLVQSGGGLVQPG QSALTQPPSVSGSPGQSVEVQLVQSGGGLVQPGRLSLRLS 111-115 SSGHNF YTWV RSLRLSCAASGFTFDTISCTGSSSDVGYYDHVS CAASGFTFDDYAMHWVRQAPGK DY DYAMHWVRQAPGKGLWYQHHPGRAPKVIIYDVT GLEWVSGISWNSGSIGYADSVK EWVSGISWNSGSIGYKRPSGVPDRFSGSKSGNT GRFTISRDNSKNTLYLQMNSLR ADSVKGRFTISRDNSASLTISGLQAEDEADYYC AEDTAVYYCAKVDGLEYSSGHN KNTLYLQMNSLRAEDSSYAGSYTWVFGGGTELT FDYWGQGTLVTVSSGSASAPTL TAVYYCAKVDGLEYS VLGGGGSGGGGSAAAQSALTQPPS SGHNFDYWGQGTLVT VSGSPGQSVTISCTGSSSDVGY VSSYDHVSWYQHHPGRAPKVIIYDV TKRPSGVPDRFSGSKSGNTASL TISGLQAEDEADYYCSSYAGSYTWVFGGGTELVTVLGQPKAAPS VTLFPPSS

In some embodiments, anti-pro/latent-myostatin antibody, orantigen-binding portion thereof, of the disclosure include any antibody,or antigen binding fragment thereof, that includes a CDRH1, CDRH2,CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, as provided forany one of the antibodies shown in Tables 1-3. In some embodiments,anti-pro/latent-myostatin antibodies, or antigen-binding portionsthereof, comprise the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 ofany one of the antibodies shown in Tables 1-3. The disclosure alsoincludes any nucleic acid sequence that encodes a molecule comprising aCDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 as provided for any one ofthe antibodies shown in Tables 1-3. Antibody heavy and light chain CDR3domains may play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen. Accordingly, theanti-pro/latent myostatin antibodies, or antigen-binding portionsthereof, of the disclosure, or the nucleic acid molecules thereof, mayinclude at least the heavy and/or light chain CDR3s of antibodies asshown in Tables 1-3.

Aspects of the disclosure relate to a monoclonal antibody, or antigenbinding fragment, that binds to pro/latent-myostatin protein and thatcomprises six complementarity determining regions (CDRs): CDRH1, CDRH2,CDRH3, CDRL1, CDRL2, and CDRL3.

In some embodiments, CDRH1 comprises a sequence as set forth in any oneof SEQ ID NOs: 1-3. In some embodiments, CDRH2 comprises a sequence asset forth in any one of SEQ ID NOs: 4-9. In some embodiments, CDRH3comprises a sequence as set forth in any one of SEQ ID NOs: 10-11, 66,71, 76, 81, 86, 91, 96, 101, 106 and 111. CDRL1 comprises a sequence asset forth in any one of SEQ ID NOs: 12-17. In some embodiments, CDRL2comprises a sequence as set forth in any one of SEQ ID NOs: 18-21. Insome embodiments, CDRL3 comprises a sequence as set forth in any one ofSEQ ID NOs: 22-23, 67, 72, 77, 82, 87, 92, 97, 102, 107 and 112.

In some embodiments (e.g., as for anti-pro/latent-myostatin antibodyAb1, shown in Table 1, or an antigen-binding portion thereof), CDRH1comprises a sequence as set forth in SEQ ID NO: 1 or 2, CDRH2 comprisesa sequence as set forth in SEQ ID NO: 4 or 5, CDRH3 comprises a sequenceas set forth in SEQ ID NO: 10, CDRL1 comprises a sequence as set forthin SEQ ID NO: 12, or 13, CDRL2 comprises a sequence as set forth in SEQID NO: 18 or 19, and CDRL3 comprises a sequence as set forth in SEQ IDNO: 22, and the antibody, or an antigen-binding portion thereof, bindsto pro/latent-myostatin.

In some embodiments (e.g., as for anti-pro/latent-myostatin antibodyAb2, shown in Table 1, or an antigen-binding portion thereof), CDRH1comprises a sequence as set forth in SEQ ID NO: 1 or 2, CDRH2 comprisesa sequence as set forth in SEQ ID NO: 4 or 5, CDRH3 comprises a sequenceas set forth in SEQ ID NO: 66, CDRL1 comprises a sequence as set forthin SEQ ID NO: 12, or 13, CDRL2 comprises a sequence as set forth in SEQID NO: 18 or 19, and CDRL3 comprises a sequence as set forth in SEQ IDNO: 67, and the antibody, or an antigen-binding portion thereof, bindsto pro/latent-myostatin.

In some embodiments (e.g., as for anti-pro/latent-myostatin antibodyAb3, shown in Table 1, or an antigen-binding portion thereof), CDRH1comprises a sequence as set forth in SEQ ID NO: 1 or 3, CDRH2 comprisesa sequence as set forth in SEQ ID NO: 6 or 7, CDRH3 comprises a sequenceas set forth in SEQ ID NO: 11, CDRL1 comprises a sequence as set forthin SEQ ID NO: 14, or 15, CDRL2 comprises a sequence as set forth in SEQID NO: 20 or 21, and CDRL3 comprises a sequence as set forth in SEQ IDNO: 23, and the antibody, or an antigen-binding portion thereof, bindsto pro/latent-myostatin.

In some embodiments (e.g., as for anti-pro/latent-myostatin antibodyAbs, shown in Table 1, or an antigen-binding portion thereof), CDRH1comprises a sequence as set forth in SEQ ID NO: 1 or 3, CDRH2 comprisesa sequence as set forth in SEQ ID NO: 8 or 9, CDRH3 comprises a sequenceas set forth in SEQ ID NO: 11, CDRL1 comprises a sequence as set forthin SEQ ID NO: 16, or 17, CDRL2 comprises a sequence as set forth in SEQID NO: 20 or 21, and CDRL3 comprises a sequence as set forth in SEQ IDNO: 23, and the antibody, or an antigen-binding portion thereof, bindsto pro/latent-myostatin.

In some examples, any of the anti-pro/latent-myostatin antibodies, orantigen-binding portions thereof, of the disclosure include any antibodyor antigen binding fragment having one or more CDR (e.g., CDRH or CDRL)sequences substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2,and/or CDRL3. For example, the antibodies may include one or more CDRsequences as shown in Tables 1-3 (SEQ ID NOs: 1-23, 66, 67, 71, 72, 76,77, 81, 82, 86, 87, 91, 92, 96, 97, 101, 102, 106, 107, 111 and 112)containing up to 5, 4, 3, 2, or 1 amino acid residue variations ascompared to the corresponding CDR region in any one of SEQ ID NOs: 1-23,66, 67, 71, 72, 76, 77, 81, 82, 86, 87, 91, 92, 96, 97, 101, 102, 106,107, 111 and 112.

In some embodiments, anti-pro/latent-myostatin antibodies, orantigen-binding portions thereof, of the disclosure include any antibodythat includes a heavy chain variable domain of any one of SEQ ID NOs:24-29, 73, 78, 83, 88, 93, 98, 103, 108 and 113 or a light chainvariable domain of any one of SEQ ID NOs: 30-35, 74, 79, 84, 89, 94, 99,104, 109 and 114. In some embodiments, anti-pro/latent-myostatinantibodies, or antigen-binding portions thereof, of the disclosureinclude any antibody that includes the heavy chain variable and lightchain variable pairs of SEQ ID NOs: 24 and 30; 25 and 31; 26 and 32; 27and 33; 28 and 34; or 29 and 35).

Aspects of the disclosure provide anti-pro/latent-myostatin antibodies,or antigen-binding portions thereof, having a heavy chain variableand/or a light chain variable amino acid sequence homologous to any ofthose described herein. In some embodiments, theanti-pro/latent-myostatin antibody, or antigen-binding portions thereof,comprises a heavy chain variable sequence or a light chain variablesequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%)identical to the heavy chain variable sequence of any of SEQ ID NOs:24-29, 73, 78, 83, 88, 93, 98, 103, 108 and 113 or a light chainvariable sequence of any one of SEQ ID NOs: 30-35, 74, 79, 84, 89, 94,99, 104, 109 and 114. In some embodiments, the homologous heavy chainvariable and/or a light chain variable amino acid sequences do not varywithin any of the CDR sequences provided herein. For example, in someembodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%,95%, 98%, or 99%) may occur within a heavy chain variable and/or a lightchain variable sequence excluding any of the CDR sequences providedherein.

The “percent identity” of two amino acid sequences is determined usingthe algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol.Biol. 215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, word length=3 to obtain amino acid sequenceshomologous to the protein molecules of interest. Where gaps existbetween two sequences, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

In some embodiments, conservative mutations can be introduced into theCDRs or framework sequences at positions where the residues are notlikely to be involved in interacting with pro/latent-myostatin asdetermined based on the crystal structure. As used herein, a“conservative amino acid substitution” refers to an amino acidsubstitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the antibodies, or antigen binding fragmentsthereof, provided herein comprise mutations that confer desirableproperties to the antibodies, or antigen binding fragments thereof. Forexample, to avoid potential complications due to Fab-arm exchange, whichis known to occur with native IgG4 mAbs, the antibodies, or antigenbinding fragments thereof, provided herein may comprise a stabilizing‘Adair’ mutation (Angal S., et al., “A single amino acid substitutionabolishes the heterogeneity of chimeric mouse/human (IgG4) antibody,”Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue241 Kabat numbering) is converted to proline resulting in an IgGl-like(CPPCP (SEQ ID NO: 58)) hinge sequence. Accordingly, any of theantibodies may include a stabilizing ‘Adair’ mutation or the amino acidsequence CPPCP (SEQ ID NO: 58).

Anti-pro/latent-myostatin antibodies, or antigen-binding portionsthereof, of this disclosure may optionally comprise antibody constantregions or parts thereof. For example, a V_(L) domain may be attached atits C-terminal end to a light chain constant domain like Cκ or Cλ.Similarly, a V_(H) domain or portion thereof may be attached to all orpart of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and any isotypesubclass. Antibodies may include suitable constant regions (see, forexample, Kabat et al., Sequences of Proteins of Immunological Interest,No. 91-3242, National Institutes of Health Publications, Bethesda, Md.(1991)). Therefore, antibodies within the scope of this may disclosureinclude V_(H) and V_(L) domains, or an antigen binding portion thereof,combined with any suitable constant regions.

In certain embodiments, the V_(H) and/or V_(L) domains may be revertedto germline sequence, e.g., the FR of these domains are mutated usingconventional molecular biology techniques to match those produced by thegermline cells. For example, the V_(H) and/or V_(L) domains may bereverted to germline sequence of IgHV3-30 (SEQ ID NO: 36) and/orIgLV1-44 (SEQ ID NO: 37), respectively. It should be appreciated thatany of the V_(H) and/or V_(L) domains may be reverted to any suitablegermline sequence. In other embodiments, the FR sequences remaindiverged from the consensus germline sequences.

IgHV3-30 (SEQ ID NO: 36)QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR IgL V1-44(SEQ ID NO: 37) QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNG

In some embodiments, anti-pro/latent-myostatin antibodies or antigenbinding fragments may or may not include the framework region of theantibodies shown in SEQ ID NOs: 24-35. In some embodiments,anti-pro-latent-myostatin antibodies are murine antibodies and includemurine framework region sequences.

In some embodiments, an anti-pro/latent-myostatin antibodies, or antigenbinding fragments thereof, can bind to pro/latent-myostatin withrelatively high affinity, e.g., with a Kd less than 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M or lower. For example,anti-pro/latent-myostatin antibodies, or antigen binding fragmentsthereof, can bind to pro/latent-myostatin with an affinity between 5 pMand 500 nM, e.g., between 50 pM and 100 nM, e.g., between 500 pM and 50nM. The invention also includes antibodies or antigen binding fragmentsthat compete with any of the antibodies described herein for binding topro/latent-myostatin and that have an affinity of 50 nM or lower (e.g.,20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower, or 5 pMor lower). The affinity and binding kinetics of theanti-pro/latent-myostatin antibody can be tested using any suitablemethod including but not limited to biosensor technology (e.g., OCTET orBIACORE). When such binding profiles are measured with the use of OCTETor BIACORE, the assay is performed typically in accordance with themanufacturer's instructions, unless otherwise specified.

In some embodiments, antibodies, or antigen binding fragments thereof,are disclosed herein that specifically bind pro/latent-myostatin. Insome embodiments, any of the antibodies, or antigen binding fragmentsthereof, provided herein bind at or near a tolloid cleavage site or ator near a tolloid docking sitc of pro/latcnt-myostatin. In someembodiments, an antibody binds near a tolloid cleavage site or near atolloid docking site if it binds within 15 or fewer amino acid residuesof the tolloid cleavage site or tolloid docking site. In someembodiments, any of the antibodies, or antigen binding fragmentsthereof, provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 amino acid residues of a tolloid cleavage site ortolloid docking site. In some embodiments, an antibody binds at or neara tolloid cleavage site of GDF8. For example, an antibody may bind anamino acid sequence as set forth in SEQ ID NO: 62PKAPPLRELIDQYDVQRDDSSDGSLEDDDYHAT (SEQ ID NO: 62). In other embodiments,any of the antibodies, or antigen binding fragments thereof, providedherein bind at or near a proprotein convertase cleavage site or at ornear a proprotein convertase docking site of pro/latent-myostatin. Insome embodiments, an antibody binds near a proprotein convertasecleavage site or near a proprotein convertase docking site if it bindswithin 15 or fewer amino acid residues of the proprotein convertasecleavage site or proprotein convertase docking site. In someembodiments, any of the antibodies, or antigen binding fragmentsthereof, provided herein bind within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 amino acid residues of a proprotein convertase cleavagesite or proprotein convertase docking site. In some embodiments, anantibody binds at or near a proprotein convertase cleavage site of GDF8.For example, an antibody may bind an amino acid sequence as set forth inSEQ ID NO: 63 (GLNPFLEV KV TDTPKRSRRDFGLDCDEHSTESRC).

In one example, the anti-pro/latent-myostatin antibodies, or antigenbinding fragments thereof, described herein specifically bindpro/latent-myostatin as compared to other forms of Myostatin and/orother members of the TGFβ family of growth factors. Members of the TGFβfamily of growth factors include, without limitation AMH, ARTN, BMP10,BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, GDF1, GDF10,GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF,INHA, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, NODAL, NRTN, PSPN,TGFβ1, TGFβ2, and TGFβ3 protein. Such antibodies, or antigen bindingfragments thereof, may bind pro/latent-myostatin at a much higheraffinity as compared to other members of the TGFβ family of growthfactors (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold,200-fold, 500-fold, or 1,000-fold higher). In some embodiments, suchantibodies, or antigen binding fragments thereof, may bindpro/latent-myostatin with an affinity of at least 000-fold higher ascompared to other members of the TGFβ family of growth factors. In someembodiments, antibodies, or antigen binding fragments thereof, providedherein may bind to pro/latent-myostatin at a much higher affinity ascompared to one or more forms of GDF11 or mature myostatin (e.g., atleast 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or1,000-fold higher). In some embodiments, antibodies, or antigen bindingfragments thereof, provided herein may bind to pro/latent-myostatin withan affinity of at least 1,000-fold higher as compared to one or moreforms of GDF11 (e.g., proGDF11, latent GDF11 or mature GDF11) or maturemyostatin. Alternatively, or in addition, antibodies, or antigen bindingfragments thereof, may exhibit a much higher inhibitory activity againstproteolytic cleavage of pro/latent-myostatin (e.g., by a proproteinconvertase or tolloid protease) as compared with other members of theTGFβ family, such as pro/latent GDF11 (e.g., at least 2-fold, 5-fold,10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold higher). Inanother embodiment, the antibodies, or antigen binding fragmentsthereof, disclosed herein do not bind to GDF11. This avoids potentialtoxicity issues associated with antibodies that cross-react with bothmyostatin and GDF11.

In some embodiments, antibodies bind an antigen but cannot effectivelyeliminate the antigen from the plasma. Thus, in some embodiments, theconcentration of the antigen in the plasma may be increased by reducingthe clearance of the antigen. However, in some embodiments, antibodies(e.g., sweeping antibodies) provided herein have an affinity to anantigen that is sensitive to pH. Such pH sensitive antibodies may bindto the antigen in plasma at neutral pH and dissociate from the antigenin an acidic endosome, thus reducing antibody-mediated antigenaccumulation and/or promoting antigen clearance from the plasma.

Aspects of the disclosure relate to sweeping antibodies. As used herein“sweeping antibodies” or antigen-binding fragments thereof refer toantibodies, or antigen-binding fragments thereof, having bothpH-sensitive antigen binding and at least a threshold level of bindingto cell surface neonatal Fc receptor (FcRn) at neutral or physiologicalpH. In some embodiments, sweeping antibodies, or an antigen-bindingportion thereof, bind to the neonatal Fc receptor FcRn at neutral pH.For example, sweeping antibodies may bind to the FcRn at a pH rangingfrom 7.0 to 7.6. In some embodiments, sweeping antibodies, or anantigen-binding portion thereof, can bind to an antigen at an antigenbinding site and bind to a cellular FcRn via an Fc portion of theantibody. In some embodiments, sweeping antibodies, or anantigen-binding portion thereof, may then be internalized, releasingantigen in an acidic endosome, which may be degraded. In someembodiments, a sweeping antibody, or an antigen-binding portion thereof,no longer bound to the antigen, may then be released (e.g., byexocytosis) by the cell back into the serum.

In some embodiments, FcRn in the vascular endothelia (e.g., of asubject) extends the half-life of a sweeping antibody, or anantigen-binding portion thereof. In some embodiments, vascularendothelial cells internalize sweeping antibodies, or antigen-bindingportions thereof, which in some embodiments are hound to an antigen suchas myostatin (e.g., pro-myostatin, latent myostatin or primedmyostatin). In some embodiments, a sweeping antibody, or anantigen-binding portion thereof, is recycled back into the bloodstream.In some embodiments, a sweeping antibody, or an antigen-binding portionthereof, has an increased half-life (e.g., in the serum of a subject) ascompared to its conventional counterpart. In some embodiments, aconventional counterpart of a sweeping antibody refers the antibody, oran antigen-binding portion thereof, from which the sweeping antibody, oran antigen-binding portion thereof, was derived (e.g., prior toengineering the Fc portion of the conventional antibody to bind FcRnwith greater affinity at pH 7). In some embodiments, a sweepingantibody, or an antigen-binding portion thereof, has a half-life in theserum of a subject that is at least 1%, 5%, 10%, 15%, 20%, 25%, 35%,50%, 75%, 100%, 150%, 200% or 250% longer as compared to itsconventional counterpart.

In some embodiments, an Fc portion of a sweeping antibody binds FcRn. Insome embodiments, the Fe portion of a sweeping antibody binds to FcRn ata pH of 7.4 with a Kd ranging from 10⁻³ M to 10⁻⁸ M. In someembodiments, a sweeping antibody binds to FcRn at a pH of 7.4 with a Kdranging from 10⁻³ M to 10⁻⁷ M, from 10⁻³ M to 10⁻⁶M, from 0⁻³ M to 10⁻⁵M, from 10⁻³ M to 10⁴ M, from 10⁻⁴M to 10⁻⁸ M, from 10⁻⁴ M to 10⁻⁷ M,from 10⁻⁴ M to 10⁻⁶ M, from 10⁻⁴ M to 10⁻⁵ M, from 10⁻⁵ M to 10⁻⁸ M,from 10⁻⁵ M to 10⁻⁷ M, from 10⁻⁵ M to 10⁻⁶ M, from 10⁻⁶ M to 10⁻⁸ M,from 10⁻⁶ M to 10⁻⁷ M, or from 10⁻⁷ M to 10⁻⁸M. In some embodiments,FcRn binds to the CH2-CH3 hinge region of a sweeping antibody. In someembodiments, FcRn binds to the same region as protein A or protein G. Insome embodiments, FcRn binds to a different binding site from FcγRs. Insome embodiments, the amino acid residues AA of a sweeping antibody Fcregion are required for binding to FcRn. In some embodiments, the aminoacid residues AA of a sweeping antibody Fc region affect binding toFcRn.

In some embodiments, any of the antibodies, or antigen binding fragmentsthereof, provided herein are engineered to bind FcRn with greateraffinity. In some embodiments, any of the antibodies, or antigen bindingfragments thereof, provided herein are engineered to bind FcRn withgreater affinity at pH 7.4. In some embodiments, the affinity ofantibodies, or antigen binding fragments thereof, to FcRn is increasedto extend their pharmacokinetic (PK) properties as compared to theirconventional counterparts. For example, in some embodiments, sweepingantibodies elicit less adverse reactions due to their efficacy at lowerdoses. In some embodiments, sweeping antibodies, or an antigen-bindingportion thereof, are administered less frequently. In some embodiments,transcytosis of sweeping antibodies, or an antigen-binding portionthereof, to certain tissue types are increased. In some embodiments,sweeping antibodies, or antigen-binding portions thereof, enhanceefficiency of trans-placental delivery. In some embodiments, sweepingantibodies, or antigen-binding portions thereof, are less costly toproduce.

In some embodiments, any of the antibodies, or antigen binding fragmentsthereof, provided herein are engineered to hind FcRn with loweraffinity. In some embodiments, any of the antibodies, or antigen bindingfragments thereof, provided herein are engineered to bind FcRn withlower affinity at pH 7.4. In some embodiments, the affinity of sweepingantibodies, or an antigen-binding portion thereof, to FcRn is decreasedto shorten their pharmacokinetic (PK) properties as compared to theirconventional counterparts. For example, in some embodiments, sweepingantibodies, or an antigen-binding portion thereof, are more rapidlycleared for imaging and/or radioimmunotherapy. In some embodiments,sweeping antibodies, or an antigen-binding portion thereof, promoteclearance of endogenous pathogenic antibodies as a treatment forautoimmune diseases. In some embodiments, sweeping antibodies, orantigen-binding portions thereof, reduce the risk of adverse pregnancyoutcome, which may be caused by trans-placental transport of materialfetus-specific antibodies.

In some embodiments, sweeping antibodies, or an antigen-binding portionthereof, have decreased affinity to an antigen at low pH as compared toa neutral or physiological pH (e.g., pH 7.4). In some embodiments,sweeping antibodies, or an antigen-binding portion thereof, have adecreased affinity to an antigen at an acidic pH (e.g. a pH ranging from5.5 to 6.5) as compared to a physiological pH (e.g., pH 7.4).

It should be appreciated that any of the antibodies, or antigen bindingfragments thereof, provided herein can be engineered to dissociate fromthe antigen depending on changes in pH (e.g., pH-sensitive antibodies).In some embodiments, sweeping antibodies, or an antigen-binding portionthereof, provided herein are engineered to bind antigen in apH-dependent manner. In some embodiments, sweeping antibodies, or anantigen-binding portion thereof, provided herein are engineered to bindFcRn in a pH-dependent manner. In soma embodiments, sweeping antibodies,or an antigen-binding portion thereof, provided herein are internalizedby endocytosis. In some embodiments, sweeping antibodies, or anantigen-binding portion thereof, provided here are internalized by FcRnbinding. In some embodiments, endocytosed sweeping antibodies, orantigen-binding portion thereof, release antigen in an endosome. In someembodiments, sweeping antibodies, or antigen-binding portions thereof,are recycled back to the cell surface. In some embodiments, sweepingantibodies remain attached to cells. In some embodiments, endocytosedsweeping antibodies, or an antigen-binding portion thereof, are recycledback to the plasma. It should be appreciated that the Fc portion of anyof the antibodies, or antigen binding fragments thereof, provided hereinmay be engineered to have different FcRn binding activity. In someembodiments, FcRn binding activity affects the clearance time of anantigen by a sweeping antibody. In some embodiments, sweeping antibodiesmay be long-acting or rapid-acting sweeping antibodies.

In some embodiments, converting a conventional therapeutic antibody, oran antigen-binding portion thereof, into a sweeping antibody, or anantigen-binding portion thereof, reduces the efficacious dose. In someembodiments, converting a conventional therapeutic antibody, or anantigen-binding portion thereof, into a sweeping antibody, or anantigen-binding portion thereof, reduces the efficacious dose by atleast 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or99%. In some embodiments, converting a conventional therapeuticantibody, or an antigen-binding portion thereof, into a sweepingantibody, or an antigen-binding portion thereof, reduces the efficaciousdose by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,8-fold, 10-fold, 15-fold, 20-fold, 50-fold or 100-fold.

In some embodiments, selecting an appropriate dose of a sweepingantibody, or an antigen-binding portion thereof, for therapy may beperformed empirically. In some embodiments, a high dose of a sweepingantibody, or an antigen-binding portion thereof, may saturate FcRn,resulting in antibodies which stabilize antigen in serum without beinginternalized. In some embodiments, a low dose of a sweeping antibody, oran antigen-binding portion thereof, may not be therapeuticallyeffective. In some embodiments, sweeping antibodies, or antigen-bindingportions thereof, are administered once a day, once a week, once everytwo weeks, once every three weeks, once every four weeks, once every 6weeks, once every 8 weeks, once every 10 weeks, once every 12 weeks,once every 16 weeks, once every 20 weeks, or once every 24 weeks.

In some embodiments, any of the antibodies, or antigen binding fragmentsthereof, provided herein may be modified or engineered to be sweepingantibodies. In some embodiments, any of the antibodies, or antigenbinding fragments thereof, provided herein may be converted into asweeping antibody using any suitable method. For example, suitablemethods for making sweeping antibodies, or antigen-binding portionsthereof, have been previously described in Igawa et al., (2013)“Engineered Monoclonal Antibody with Novel Antigen-Sweeping Activity Invivo,” PLoS ONE 8(5): e63236; and Igawa et al., “pH-dependentantigen-binding antibodies as a novel therapeutic modality,” Biochimicaet Biophysica Acta 1844 (2014) 1943-1950; the contents of each of whichare hereby incorporated by reference. It should be appreciated, however,that the methods for making sweeping antibodies, or an antigen-bindingportion thereof, as provided herein are not meant to be limiting. Thus,additional methods for making sweeping antibodies, or an antigen-bindingportion thereof, are within the scope of this disclosure.

Some aspects of the disclosure are based on the recognition that theaffinity (e.g., as expressed as Kd) of any of theanti-pro/latent-myostatin antibodies, or antigen binding fragmentsthereof, provided herein are sensitive to changes in pH. In someembodiments, the antibodies, or antigen binding fragments thereof,provided herein have an increased Kd of binding to pro/latent-myostatinat a relatively low pH (e.g., a pH ranging from 4.0-6.5) as compared toa relatively high pH (e.g., a pH ranging from 7.0-7.4). In someembodiments, the antibodies, or antigen binding fragments thereof,provided herein have a Kd of binding to pro/latent-myostatin rangingfrom 10⁻³ M, 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M when the pH isbetween 4.0 and 6.5. In some embodiments, the antibodies, or antigenbinding fragments thereof, provided herein have a Kd of binding topro/latent-myostatin ranging from 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰M, 10¹¹ M when the pH is between 7.0 and 7.4. In some embodiments, theantibodies, or antigen binding fragments thereof, provided herein have aKd of binding to pro/latent-Myostatin that is at least 2-fold, at least10-fold, at least 50-fold, at least 100-fold, at least 500-fold, atleast 1000-fold, at least 5000-fold, or at least 10000-fold greater at apH between 4.0 and 6.5 as compared to a pH between 7.0 and 7.4.

In some embodiments, pro/latent-myostatin antibodies, or antigen bindingfragments thereof, are provided herein that do not specifically bind toan epitope within the amino acid sequence set forth as (SEQ ID NO: 64).In some embodiments, pro/latent-myostatin antibodies, or antigen bindingfragments thereof, provided herein do not specifically bind to the sameepitope as an antibody described in Table 2a, 11a, 11b, or 13 ofInternational Patent Application Publication No. WO 2016/098357, whichwas published on Jun. 23, 2016, and which is based on InternationalPatent Application No. PCT/JP2015/006323, which was filed on Dec. 18,2015. In some embodiments, pro/latent-myostatin antibodies, or antigenbinding fragments thereof, provided herein do not compete or do notcross-compete for binding to the same epitope as an antibody describedin Table 2a, 11a, 11b, or 13 of International Patent ApplicationPublication No. WO 2016/098357, which was published on Jun. 23, 2016,and which is based on International Patent Application No.PCT/JP2015/006323, which was filed on Dec. 18, 2015. In someembodiments, pro/latent-myostatin antibodies, or antigen bindingfragments thereof, provided herein do not specifically bind to the sameepitope as an antibody comprising a VH and a VL pair described in Table2a, 11a, 1 lb, or 13 of International Patent Application Publication No.WO 2016/098357, which was published on Jun. 23, 2016, and which is basedon International Patent Application No. PCT/JP2015/006323, which wasfiled on Dec. 18, 2015. In some embodiments, pro/latent-myostatinantibodies, or antigen binding fragments thereof, provided herein do notcompete or do not cross-compete for binding to the same epitope as anantibody comprising a VH and a VL pair described in Table 2a, 11a, 11b,or 13 of International Patent Application Publication No. WO2016/098357, which was published on Jun. 23, 2016, and which is based onInternational Patent Application No. PCT/J P2015/006323, which was filedon Dec. 18, 2015.

Polypeptides

Some aspects of the disclosure relate to a polypeptide having a sequenceselected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO 29. In someembodiments, the polypeptide is a variable heavy chain domain. In someembodiments, the polypeptide is at least 75% (e.g., 80%, 85%, 90%, 95%,98%, or 99%) identical to any one of the amino acid sequences set forthin SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ IDNO: 28, or SEQ ID NO 29.

Some aspects of the disclosure relate to a polypeptide having a sequenceselected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO 35. In someembodiments, the polypeptide is a variable light chain domain. In someembodiments, the polypeptide is at least 75% (e.g., 80%, 85%, 90%, 95%,98%, or 99%) identical to any one of the amino acid sequences set forthin SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ IDNO: 34, or SEQ ID NO 35.

Antibodies, and Antigen-Binding Fragments, that Compete withAnti-Pro/Latent-Myostatin Antibodies, or Antigen Binding FragmentsThereof

Aspects of the disclosure relate to antibodies, and antigen-bindingfragments thereof, that compete or cross-compete with any of theantibodies, or antigen binding fragments thereof, provided herein. Theterm “compete”, as used herein with regard to an antibody, means that afirst antibody binds to an epitope of a protein (e.g., latent myostatin)in a manner sufficiently similar to the binding of a second antibody,such that the result of binding of the first antibody with its epitopeis detectably decreased in the presence of the second antibody comparedto the binding of the first antibody in the absence of the secondantibody. The alternative, where the binding of the second antibody toits epitope is also detectably decreased in the presence of the firstantibody, can, but need not be the case. That is, a first antibody caninhibit the binding of a second antibody to its epitope without thatsecond antibody inhibiting the binding of the first antibody to itsrespective epitope. However, where each antibody detectably inhibits thebinding of the other antibody with its epitope or ligand, whether to thesame, greater, or lesser extent, the antibodies are said to“cross-compete” with each other for binding of their respectiveepitope(s). Both competing and cross-competing antibodies are within thescope of this disclosure. Regardless of the mechanism by which suchcompetition or cross-competition occurs (e.g., steric hindrance,conformational change, or binding to a common epitope, or portionthereof), the skilled artisan would appreciate that such competingand/or cross-competing antibodies are encompassed and can be useful forthe methods and/or compositions provided herein.

Aspects of the disclosure relate to antibodies, or antigen-bindingportions thereof, that compete or cross-compete with any of theantibodies, or antigen binding fragments thereof, provided herein. Insome embodiments, an antibody, or an antigen-binding portion thereof,binds at or near the same epitope as any of the antibodies providedherein. In some embodiments, an antibody, or an antigen-binding portionthereof, binds near an epitope if it binds within 15 or fewer amino acidresidues of the epitope. In some embodiments, any of the antibodies, orantigen binding fragments thereof, provided herein bind within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues of anepitope that is bound by any of the antibodies, or antigen bindingfragments thereof, provided herein.

In another embodiment, an antibody, or an antigen-binding portionthereof, competes or cross-competes for binding to any of the antigensprovided herein (e.g., pro/latent-myostatin) with an equilibriumdissociation constant, Kd, between the antibody and the protein of lessthan 10⁻⁶ M. In other embodiments, an antibody, or an antigen-bindingportion thereof, competes or cross-competes for binding to any of theantigens provided herein with a Kd in a range from 10⁻¹¹ M to 10⁻⁶ M.

Aspects of the disclosure relate to antibodies, or antigen-bindingportions thereof, that compete for binding to pro/latent-myostatin withany of the antibodies, or antigen binding fragments thereof, providedherein. In some embodiments, the antibody, or an antigen-binding portionthereof, binds to pro/latent-myostatin at the same epitope as any of theantibodies, or antigen-binding portions thereof, provided herein. Forexample, in some embodiments any of the antibodies provided herein bindat or near a tolloid cleavage site or at or near a tolloid docking siteof pro/latent-myostatin. In other embodiments, any of the antibodies, orantigen binding fragments thereof, provided herein bind at or near aproprotein convertase cleavage site or at or near a proproteinconvertase docking site of pro/latent-myostatin. In another embodiment,an antibody, or an antigen-binding portion thereof, competes for bindingto pro/latent-myostatin with an equilibrium dissociation constant, Kd,between the antibody, or antigen-binding portion thereof, andpro/latent-myostatin of less than 10⁻⁶ M. In other embodiments, theantibody, or antigen-binding portion thereof, that competes with any ofthe antibodies, or antigen-binding portions thereof, provided hereinbinds to pro/latent-myostatin with a Kd in ranging from 10⁻¹¹ M to 10⁻⁶M.

Any of the antibodies, or antigen binding fragments thereof, providedherein can be characterized using any suitable methods. For example, onemethod is to identify the epitope to which the antigen binds, or“epitope mapping.” There are many suitable methods for mapping andcharacterizing the location of epitopes on proteins, including solvingthe crystal structure of an antibody-antigen complex, competitionassays, gene fragment expression assays, and synthetic peptide-basedassays, as described, for example, in Chapter 11 of Harlow and Lane,Using Antibodies, a Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1999. In an additional example, epitopemapping can be used to determine the sequence to which an antibody, oran antigen-binding portion thereof, binds. The epitope can be a linearepitope, i.e., contained in a single stretch of amino acids, or aconformational epitope formed by a three-dimensional interaction ofamino acids that may not necessarily be contained in a single stretch(primary structure linear sequence). Peptides of varying lengths (e.g.,at least 4-6 amino acids long) can be isolated or synthesized (e.g.,recombinantly) and used for binding assays with an antibody. In anotherexample, the epitope to which the antibody, or an antigen-bindingportion thereof, binds can be determined in a systematic screen by usingoverlapping peptides derived from the target antigen sequence anddetermining binding by the antibody, or an antigen-binding portionthereof. According to the gene fragment expression assays, the openreading frame encoding the target antigen is fragmented either randomlyor by specific genetic constructions and the reactivity of the expressedfragments of the antigen with the antibody to be tested is determined.The gene fragments may, for example, be produced by PCR and thentranscribed and translated into protein in vitro, in the presence ofradioactive amino acids. The binding of the antibody, or anantigen-binding portion thereof, to the radioactively labeled antigenfragments is then determined by immunoprecipitation and gelelectrophoresis. Certain epitopes can also be identified by using largelibraries of random peptide sequences displayed on the surface of phageparticles (phage libraries). Alternatively, a defined library ofoverlapping peptide fragments can be tested for binding to the testantibody, or an antigen-binding portion thereof, in simple bindingassays. In an additional example, mutagenesis of an antigen bindingdomain, domain swapping experiments and alanine scanning mutagenesis canbe performed to identify residues required, sufficient, and/or necessaryfor epitope binding. For example, domain swapping experiments can beperformed using a mutant of a target antigen in which various fragmentsof the pro/latent-myostatin polypeptide have been replaced (swapped)with sequences from a closely related, but antigenically distinctprotein, such as another member of the TGFβ protein family (e.g.,GDF11). By assessing binding of the antibody, or antigen-binding portionthereof, to the mutant pro/latent-myostatin, the importance of theparticular antigen fragment to antibody, or antigen-binding portionthereof, binding can be assessed.

Alternatively, competition assays can be performed using otherantibodies known to bind to the same antigen to determine whether anantibody, or an antigen-binding portion thereof, binds to the sameepitope as the other antibodies, or antigen-binding portions thereof.Competition assays are well known to those of skill in the art.

Any of the suitable methods, e.g., the epitope mapping methods asdescribed herein, can be applied to determine whether ananti-pro/latent-myostatin antibody, or an antigen-binding portionthereof, binds one or more of the specific residues/segments inpro/latent-myostatin as described herein. Further, the interaction ofthe antibody, or an antigen-binding portion thereof, with one or more ofthose defined residues in pro/latent-myostatin can be determined byroutine technology. For example, a crystal structure can be determined,and the distances between the residues in pro/latent-myostatin and oneor more residues in the antibody, or antigen-binding portion thereof,can be determined accordingly. Based on such distance, whether aspecific residue in pro/latent-myostatin interacts with one or moreresidues in the antibody, or antigen-binding portion thereof, can bedetermined. Further, suitable methods, such as competition assays andtarget mutagenesis assays can be applied to determine the preferentialbinding of a candidate anti-pro/latent-myostatin antibody, or nantigen-binding portion thereof, to pro/latent-myostatin as compared toanother target such as a mutant pro/latent-myostatin.

Production of Anti-Pro/Latent-Myostatin Antibodies or Antigen BindingFragments Thereof

Numerous methods may be used for obtaining antibodies, or antigenbinding fragments thereof, of the disclosure. For example, antibodies,and antigen-binding fragments thereof, can be produced using recombinantDNA methods. Monoclonal antibodies, and antigen-binding fragmentsthereof, may also be produced by generation of hybridomas (see e.g.,Kohler and Milstein (1975) Nature, 256: 495-499) in accordance withknown methods. Hybridomas formed in this manner are then screened usingstandard methods, such as enzyme-linked immunosorbent assay (ELISA) andsurface plasmon resonance (e.g., OCTET or BIACORE) analysis, to identifyone or more hybridomas that produce an antibody, or an antigen-bindingportion thereof, that specifically binds to a specified antigen. Anyform of the specified antigen may be used as the immunogen, e.g.,recombinant antigen, naturally occurring forms, any variants orfragments thereof, as well as antigenic peptide thereof (e.g., any ofthe epitopes described herein as a linear epitope or within a scaffoldas a conformational epitope). One exemplary method of making antibodies,and antigen-binding portions thereof, includes screening proteinexpression libraries that express antibodies or fragments thereof (e.g.,scFv), e.g., phage or ribosome display libraries. Phage display isdescribed, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith(1985) Science 228:1315-1317; Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597; WO92/18619; WO91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO92/09690; and WO 90/02809.

In addition to the use of display libraries, the specified antigen(e.g., pro-myostatin) can be used to immunize a non-human animal, e.g.,a rodent, e.g., a mouse, hamster, or rat. In one embodiment, thenon-human animal is a mouse.

In another embodiment, a monoclonal antibody is obtained from thenon-human animal, and then modified, e.g., chimeric, using suitablerecombinant DNA techniques. A variety of approaches for making chimericantibodies have been described. See e.g., Morrison et al., Proc. Natl.Acad. Sci. U.S.A. 81:6851, 1985; Takeda et al., Nature 314:452, 1985,Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No.4,816,397; Tanaguchi et al., European Patent Publication EP171496;European Patent Publication 0173494, United Kingdom Patent GB 2177096B.

For additional antibody production techniques, see Antibodies: ALaboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory,1988. The present disclosure is not necessarily limited to anyparticular source, method of production, or other specialcharacteristics of an antibody.

Some aspects of the present disclosure relate to host cells transformedwith a polynucleotide or vector. Host cells may be a prokaryotic oreukaryotic cell. The polynucleotide or vector which is present in thehost cell may either be integrated into the genome of the host cell orit may be maintained extrachromosomally. The host cell can be anyprokaryotic or eukaryotic cell, such as a bacterial, insect, fungal,plant, animal or human cell. In some embodiments, fungal cells are, forexample, those of the genus Saccharomyces, in particular those of thespecies S. cerevisiae. The term “prokaryotic” includes all bacteriawhich can be transformed or transfected with a DNA or RNA molecules forthe expression of an antibody or the corresponding immunoglobulinchains. Prokaryotic hosts may include gram negative as well as grampositive bacteria such as, for example, E. coli, S. typhimurium,Serratia marcescens and Bacillus subtilis. The term “eukaryotic”includes yeast, higher plants, insects and vertebrate cells, e.g.,mammalian cells, such as NSO and CHO cells. Depending upon the hostemployed in a recombinant production procedure, the antibodies orimmunoglobulin chains encoded by the polynucleotide may be glycosylatedor may be non-glycosylated. Antibodies or the correspondingimmunoglobulin chains may also include an initial methionine amino acidresidue.

In some embodiments, once a vector has been incorporated into anappropriate host, the host may be maintained under conditions suitablefor high level expression of the nucleotide sequences, and, as desired,the collection and purification of the immunoglobulin light chains,heavy chains, light/heavy chain dimers or intact antibodies, antigenbinding fragments or other immunoglobulin forms may follow; see,Beychok, Cells of Immunoglobulin Synthesis, Academic Press, N.Y.,(1979). Thus, polynucleotides or vectors are introduced into the cellswhich in turn produce the antibody or antigen binding fragments.Furthermore, transgenic animals, preferably mammals, comprising theaforementioned host cells may be used for the large scale production ofthe antibody or antibody fragments.

The transformed host cells can be grown in fermenters and cultured usingany suitable techniques to achieve optimal cell growth. Once expressed,the whole antibodies, their dimers, individual light and heavy chains,other immunoglobulin forms, or antigen binding fragments, can bepurified according to standard procedures of the art, including ammoniumsulfate precipitation, affinity columns, column chromatography, gelelectrophoresis and the like; see, Scopes, “Protein Purification”,Springer Verlag, N.Y. (1982). The antibody or antigen binding fragmentscan then be isolated from the growth medium, cellular lysates, orcellular membrane fractions. The isolation and purification of the,e.g., microbially expressed antibodies or antigen binding fragments maybe by any conventional means such as, for example, preparativechromatographic separations and immunological separations such as thoseinvolving the use of monoclonal or polyclonal antibodies directed, e.g.,against the constant region of the antibody.

Aspects of the disclosure relate to a hybridoma, which provides anindefinitely prolonged source of monoclonal antibodies. As analternative to obtaining immunoglobulins directly from the culture ofhybridomas, immortalized hybridoma cells can be used as a source ofrearranged heavy chain and light chain loci for subsequent expressionand/or genetic manipulation. Rearranged antibody genes can be reversetranscribed from appropriate mRNAs to produce cDNA. In some embodiments,heavy chain constant region can be exchanged for that of a differentisotype or eliminated altogether. The variable regions can be linked toencode single chain Fv regions. Multiple Fv regions can be linked toconfer binding ability to more than one target or chimcric heavy andlight chain combinations can be employed. Any appropriate method may beused for cloning of antibody variable regions and generation ofrecombinant antibodies, and antigen-binding portions thereof.

In some embodiments, an appropriate nucleic acid that encodes variableregions of a heavy and/or light chain is obtained and inserted into anexpression vectors which can be transfected into standard recombinanthost cells. A variety of such host cells may be used. In someembodiments, mammalian host cells may be advantageous for efficientprocessing and production. Typical mammalian cell lines useful for thispurpose include CHO cells, 293 cells, or NSO cells. The production ofthe antibody or antigen binding fragment may be undertaken by culturinga modified recombinant host under culture conditions appropriate for thegrowth of the host cells and the expression of the coding sequences. Theantibodies or antigen binding fragments may be recovered by isolatingthem from the culture. The expression systems may be designed to includesignal peptides so that the resulting antibodies are secreted into themedium; however, intracellular production is also possible.

The disclosure also includes a polynucleotide encoding at least avariable region of an immunoglobulin chain of the antibodies describedherein. In some embodiments, the variable region encoded by thepolynucleotide comprises at least one complementarity determining region(CDR) of the VH and/or VL of the variable region of the antibodyproduced by any one of the above described hybridomas.

Polynucleotides encoding antibody or antigen binding fragments may be,e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or arecombinantly produced chimeric nucleic acid molecule comprising any ofthose polynucleotides either alone or in combination. In someembodiments, a polynucleotide is part of a vector. Such vectors maycomprise further genes such as marker genes which allow for theselection of the vector in a suitable host cell and under suitableconditions.

In some embodiments, a polynucleotide is operatively linked toexpression control sequences allowing expression in prokaryotic oreukaryotic cells. Expression of the polynucleotide comprisestranscription of the polynucleotide into a translatable mRNA. Regulatoryelements ensuring expression in eukaryotic cells, preferably mammaliancells, are well known to those skilled in the art. They may includeregulatory sequences that facilitate initiation of transcription andoptionally poly-A signals that facilitate termination of transcriptionand stabilization of the transcript. Additional regulatory elements mayinclude transcriptional as well as translational enhancers, and/ornaturally associated or heterologous promoter regions. Possibleregulatory elements permitting expression in prokaryotic host cellsinclude, e.g., the PL, Lac, Trp or Tac promoter in E. coli, and examplesof regulatory elements permitting expression in eukaryotic host cellsare the AOX1 or GAL1 promoter in yeast or the CMV-promoter,SV40-promoter, RSV-promoter (Rous sarcoma virus), CMV-enhancer,SV40-enhancer or a globin intron in mammalian and other animal cells.

Beside elements which are responsible for the initiation oftranscription such regulatory elements may also include transcriptiontermination signals, such as the SV40-poly-A site or the tk-poly-A site,downstream of the polynucleotide. Furthermore, depending on theexpression system employed, leader sequences capable of directing thepolypeptide to a cellular compartment or secreting it into the mediummay be added to the coding sequence of the polynucleotide and have beendescribed previously. The leader sequence(s) is (are) assembled inappropriate phase with translation, initiation and terminationsequences, and preferably, a leader sequence capable of directingsecretion of translated protein, or a portion thereof, into, forexample, the extracellular medium. Optionally, a heterologouspolynucleotide sequence can be used that encode a fusion proteinincluding a C- or N-terminal identification peptide imparting desiredcharacteristics, e.g., stabilization or simplified purification ofexpressed recombinant product.

In some embodiments, polynucleotides encoding at least the variabledomain of the light and/or heavy chain may encode the variable domainsof both immunoglobulin chains or only one. Likewise, a polynucleotide(s)may be under the control of the same promoter or may be separatelycontrolled for expression. Furthermore, some aspects relate to vectors,particularly plasmids, cosmids, viruses and bacteriophages usedconventionally in genetic engineering that comprise a polynucleotideencoding a variable domain of an immunoglobulin chain of an antibody orantigen binding fragment; optionally in combination with apolynucleotide that encodes the variable domain of the otherimmunoglobulin chain of the antibody.

In some embodiments, expression control sequences are provided aseukaryotic promoter systems in vectors capable of transforming ortransfecting eukaryotic host cells, but control sequences forprokaryotic hosts may also be used. Expression vectors derived fromviruses such as retroviruses, vaccinia virus, adeno-associated virus,herpes viruses, or bovine papilloma virus, may be used for delivery ofthe polynucleotides or vector into targeted cell population (e.g., toengineer a cell to express an antibody or antigen binding fragment). Avariety of appropriate methods can be used to construct recombinantviral vectors. In some embodiments, polynucleotides and vectors can bereconstituted into liposomes for delivery to target cells. The vectorscontaining the polynucleotides (e.g., the heavy and/or light variabledomain(s) of the immunoglobulin chains encoding sequences and expressioncontrol sequences) can be transferred into the host cell by suitablemethods, which vary depending on the type of cellular host.

Modifications

Antibodies and antigen binding fragments of the disclosure may bemodified with a detectable label, including, but not limited to, anenzyme, prosthetic group, fluorescent material, luminescent material,bioluminescent material, radioactive material, positron emitting metal,nonradioactive paramagnetic metal ion, and affinity label for detectionand isolation of pro/latent-myostatin. The detectable substance may becoupled or conjugated either directly to the polypeptides of thedisclosure or indirectly, through an intermediate (such as, for example,a linker) using suitable techniques. Non-limiting examples of suitableenzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, glucose oxidase, or acetylcholinesterase; non-limitingexamples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; non-limiting examples of suitablefluorescent materials include biotin, umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride, or phycoerythrin; an example of aluminescent material includes luminol; non-limiting examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include a radioactivemetal ion, e.g., alpha-emitters or other radioisotopes such as, forexample, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹⁵mIn, ¹¹³mIn, ¹¹²In, ¹¹¹In), and technetium(⁹⁹Tc, ⁹⁹mTc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, Lu,¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ⁸⁶R, ¹⁸⁸Re, ¹⁴², ¹⁰⁵Rh,⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, andtin (¹¹³Sn, ¹¹⁷Sn). The detectable substance may be coupled orconjugated either directly to the anti-pro/latent-myostatin antibodies,or antigen-binding portions thereof, of the disclosure or indirectly,through an intermediate (such as, for example, a linker) using suitabletechniques. Anti-pro/latent-myostatin antibodies, or antigen-bindingportions thereof, conjugated to a detectable substance may be used fordiagnostic assays as described herein.

Biological Effects of Myostatin Inhibitors, Such as Anti-Pro/LatentMyostatin Antibodies and Antigen Binding Fragments Thereof

Myostatin inhibitors, such as antibodies and antigen-binding fragmentsthereof, which are encompassed by the present disclosure can be used asa medicament to effectuate beneficial effects (e.g., therapeuticeffects) in a subject when administered to the subject in an effectiveamount. Exemplary such biologically beneficial effects are providedherein. Beneficial biological effects in a subject can be achieved byadministration of myostatin inhibitors, e.g., antibodies, or antigenbinding fragments thereof, as described herein, that specifically bindpro/latent myostatin. In some embodiments, the antibody, orantigen-binding portion thereof, is administered in an amount effectiveto cause two or more of the biological effects described below. In someembodiments, the myostatin inhibitor, e.g., antibody, or antigen-bindingportion thereof, is administered in an amount effective to cause threeor more of the biological effects described below. In some embodiments,the myostatin inhibitor, e.g., the antibody, or antigen-binding portionthereof, is administered in an amount effective to cause four or more ofthe biological effects described below. In some embodiments, themyostatin inhibitor, e.g., the antibody, or antigen-binding portionthereof, is administered in an amount effective to cause five or more ofthe biological effects described below. In some embodiments, themyostatin inhibitor, e.g., the antibody, or antigen-binding portionthereof, is administered in an amount effective to cause six or more ofthe biological effects described below. In some embodiments, themyostatin inhibitor, e.g., the antibody, or antigen-binding portionthereof, is administered in an amount effective to cause seven or moreof the biological effects described below. In some embodiments, themyostatin inhibitor, e.g., the antibody, or antigen-binding portionthereof, is administered in an amount effective to cause eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen of thebiological effects described below.

A. Effect on Mass and/or Function of Muscle Tissue in the Human Subject

Administration of a myostatin inhibitor, e.g., an antibody, or antigenbinding fragment thereof, that specifically binds pro/latent myostatinincreases mass and/or function of a muscle tissue in the human subject.In some embodiments, the muscle tissue is selected from the groupconsisting of a smooth muscle tissue, a skeletal muscle tissue and acardiac muscle tissue. Smooth muscle tissue is made up from longtapering cells, generally involuntary and differs from striated musclein the much higher actin/myosin ratio, the absence of conspicuoussarcomeres and the ability to contract to a much smaller fraction of itsresting length. Smooth muscle cells are found particularly in bloodvessel walls, surrounding the intestine and in the uterus. Cardiacmuscle tissue is a striated but involuntary tissue responsible for thepumping activity of the vertebrate heart. The individual cardiac musclecells are not fused together into multinucleate structures as they arein striated muscle tissue. Skeletal muscle tissue is under voluntarycontrol. The muscle fibers are syncytial and contain myofibrils, tandemarrays of sarcomeres. There are two general types of skeletal musclefibers: slow-twitch (type I) and fast-twitch (type II) according to theexpression of their particular myosin heavy chain (MHC) isoform.Slow-twitch muscles are better equipped to work aerobically and helpenable long-endurance feats such as distance running, while fast-twitchmuscles fatigue faster but are better equipped to work anaerobically andare used in powerful bursts of movements like sprinting. Thedifferentiation between slow and fast twitch muscle fibers is based onhistochemical staining for myosin adenosine-triphosphatase (ATPase) andthe type of myosin heavy chain. The slow twitch muscle fiber (type Ifiber) is MHC isoform I and the three fast twitch isoforms (type IIfibers) are MHC isoform IIa, MHC isoform IId, and MHC isoform IIb (S.Schiaffino, J. Muscle Res. Cell. Motil., 10 (1989), pp. 197-205). Insome embodiments, the mass and/or function of a fast twitch muscletissue in the human subject is increased. In other embodiments, the massand/or function of a slow twitch muscle tissue in the human subject isincreased.

Biological effects of an effective amount of the pharmaceuticalcompositions provided herein may be associated with a phenotypic changeof muscle fiber types, which is a process referred to as fiber typeswitch. In some embodiments, fiber type switch is triggered by an event,such as an injury and starvation.

In one aspect, the disclosure provides a method for promoting fiber typeswitch in a subject. The method comprises administering to the subject acomposition comprising a myostatin inhibitor, e.g., an antibody, orantigen binding fragment thereof, that specifically bindspro/latent-myostatin and blocks release of mature myostatin in an amounteffective to promote fiber type switch, thereby promoting fiber typeswitch in the subject.

In another aspect, the disclosure provides a method for preferentiallyincreasing type II or fast twitch fibers over type I or slow twitchfibers in a subject. The method comprises administering to the subject acomposition comprising a myostatin inhibitor, e.g., an antibody, orantigen binding fragment thereof, that specifically bindspro/latent-myostatin and blocks release of mature myostatin in an amounteffective to preferentially increase type II or fast twitch fibers overtype I or slow twitch fibers fiber type switch, thereby preferentiallyincreasing type II or fast twitch fibers over type I or slow twitchfibers in the subject.

In some embodiments, administration of an effective amount of amyostatin inhibitor, e.g., an antibody, or antigen-binding fragmentthereof, described herein to a subject can cause an increase in musclemass. Preferably, such an increase in muscle mass is clinicallymeaningful to benefit or otherwise improve the health status of thesubject. For example, clinically meaningful changes in muscle mass mayimprove the patient's mobility, self-care, metabolism, etc. In someembodiments, the increase in muscle mass is an increase in lean muscleor lean muscles. In some embodiments, such increase in muscle mass is asystemic effect such that muscles in the whole body or substantiallywhole body show the measurable effect. In other embodiments, effects arelocalized to certain group/type of muscles. In some embodiments, themass of the muscle tissue, e.g., lean muscle tissue, is increased by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, themass of the muscle tissue, e.g., lean muscle tissue, is increased by atleast about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%,20-60%, 30-80%, 40-90%, or 50-100%. Such increase in muscle mass may bededuced or measured by any suitable known methods, including measurementof cross-sectional area via MRI (e.g., forearm cross section),circumference, diaphragm width (e.g., via ultrasound), etc.

In some embodiments, administration of an effective amount of anantibody or antigen-binding fragments thereof described herein to asubject can cause an enhancement in muscle function. Muscle function maybe assessed by a variety of measures, including, without limitation:force generation, grip strength (e.g., maximum grip strength),endurance, muscle oxidative capacity, dynamic grip endurance, etc. Insome embodiments, serum creatinine levels are used as a validatedbiomarker indicative of muscle mass, albeit with limited sensitivity.

In some embodiments, the function of the muscle tissue is increased byat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments,the function of the muscle tissue is increased by at least about 1-5%,5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%,40-90%, or 50-100%. In some embodiments, increased muscle functioncomprises improved rating, for example, from 1 to 2, 2 to 3, 3 to 4, 4to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

In some embodiments, the myostatin inhibitors, e.g., anti-pro/latentmyostatin antibodies, or antigen binding fragments thereof, for use inthe methods of the present invention may increase the mass and/orfunction of the muscle tissue in the subject suffering from a lesion,e.g., due to a spinal cord injury. In some embodiments, the subject isin an acute spinal cord injury phase immediately after injury, wherediagnosis between complete and incomplete injury is generally difficult.In other embodiments, the subject is in a sub-acute spinal cord injuryphase, where there is a distinction between complete and incompletespinal cord injury, and recovery is possible through ongoing rehab. Inyet another embodiment, the subject is in a chronic spinal cord injuryphase. The chronic SCI phase occurs around 4 or 6 month from the date ofinjury, where patients have demonstrated substantial decrease in rate ofrecovery or when rehab efforts have reached a plateau despite theongoing standard of care efforts.

In some embodiments, the mass and/or function of the muscle tissue belowa lesion is increased in a subject suffering from a lesion, e.g., aspinal cord injury. In other embodiments, the mass and/or function ofthe muscle tissue above a lesion is increased in a subject sufferingfrom a lesion, e.g., a spinal cord injury. In some embodiment, themuscle is selected from the group consisting of a soleus muscle, agastrocnemius muscle, a bicep muscle and a tricep muscle. In someembodiments, the mass of the muscle tissue is increased by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, the mass of themuscle tissue is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%,1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. Insome embodiments, the function of the muscle tissue is increased by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, thefunction of the muscle tissue is increased by at least about 1-5%,5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%,40-90%, or 50-100%.

In some embodiments, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, that specifically bindspro/latent myostatin increases locomotor function in the human subject,e.g., in a subject suffering from a lesion. In some embodiments, thelocomotor function of the human subject is increased by at least 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,60%, 70%, 80%, 90% or 100%. In other embodiments, the locomotor functionof the human subject is increased by at least about 1-5%, 5-10%, 10-20%,1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

In some embodiments, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, that specifically bindspro/latent myostatin increases the motor ordination and balance in thehuman subject, e.g., in a subject suffering from a lesion. In someembodiments, the motor ordination and balance of the human subject isincreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In otherembodiments, the motor ordination and balance of the human subject isincreased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%,10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

In another embodiment, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, that specifically bindspro/latent myostatin increases the muscle strength in the human subject,e.g., in a subject suffering from a lesion. In some embodiments, themuscle strength of the human subject is increased by at least 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,60%, 70%, 80%, 90% or 100%. In other embodiments, the muscle strength ofthe human subject is increased by at least about 1-5%, 5-10%, 10-20%,1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

In some embodiments, administration of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, that specifically bindspro/latent myostatin can cause clinically meaningful changes in musclefunction which corresponds to enhanced functionality of the patient. Insome embodiments, enhanced functionality includes improvement in thepatient's mobility, self-care, metabolism, etc. In some embodiments,administration of an effective amount of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, that specifically bindspro/latent myostatin facilitates or accelerates recovery from acondition, such as injuries, surgeries and other medical procedures.Suitable such conditions may involve a condition that is associated witha nerve damage (whether resulting from an injury or a surgical or otherclinical procedure).

For example, suitable subjects include generally healthy individuals,such as a patient who: i) has sustained an acute injury involving anerve damage that affects muscle function; ii) is scheduled to undergo asurgical procedure (therapeutic or corrective) that may cause anunintended nerve injury (e.g., motor neuron injury); iii) has undergonea surgical procedure that has caused an unintended muscle dysfunction;iv) receives a treatment that involves immobilization of a particularmuscle or muscle groups (e.g., cast, etc.); v) is on ventilator (e.g.,as a result of acute injury). The administration of the myostatininhibitor described herein may accelerate recovery in such patients. Insome embodiments, such administration may be prophylactic. For example,prior to undergoing or immediately following a surgical procedure thatmay cause a nerve damage and associated muscle dysfunction, the antibodymay be administered to prevent muscle dysfunction. Prevention includesalleviating or lessening the severity of such dysfunction. In theseembodiments, administration may be a local administration at or near thesite of the affected area, e.g., injury, surgery, etc.

B. Effect on the Metabolic Rate of the Human Subject

Administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, that specifically binds pro/latent myostatinincreases the metabolic rate of the human subject. In some embodiments,administration of an effective amount of such myostatin inhibitor, e.g.,antibody, or antigen-binding fragment thereof, can increase the basalmetabolic rate in the subject. Metabolic rates can be calculated by anymethods known in the art, for example, by examining the oxygen input andcarbon dioxide output, or by indirect calorimetry as demonstrated byExample 11 of the present application. In some embodiments, themetabolic rate is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or100%. In other embodiments, the metabolic rate is increased by at leastabout 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%,30-80%, 40-90%, or 50-100%.

C. Effect on Insulin Sensitivity of the Human Subject

Administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, that specifically binds pro/latent myostatinincreases insulin sensitivity of the human subject. Methods formeasuring insulin sensitivity are known in the art, for example, glucosetolerance test, and fasting insulin or glucose test. During a glucosetolerance test, a fasting patient takes a 75-gram oral dose of glucose,and then blood glucose levels are measured over the following two hours.A glycemia less than 7.8 mmol/L (140 mg/dl) is considered normal, aglycemia of between 7.8 and 11.0 mmol/L (140 to 197 mg/dl) is consideredas impaired glucose tolerance (TGT), and a glycemia of greater than orequal to 11.1 mmol/L (200 mg/dl) is considered diabetes mellitus. Forfasting insulin test, a fasting serum insulin level greater than 25mIU/L or 174 pmol/L is considered insulin resistance. In someembodiments, the metabolic rate is increased by at least 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,70%, 80%, 90% or 100%. In other embodiments, the metabolic rate isincreased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%,10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

D. Effect on the Level of Adipose Tissue in the Human Subject

Administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, that specifically binds pro/latent myostatinaffects the level of adipose tissue in the human subject. As usedherein, the term “adipose tissue” refers to fat including the connectivetissue that stores fat. Adipose tissue is derived from preadipocytes.Its main role is to store energy in the form of lipids, although it alsocushions and insulates the body. The two types of adipose tissue arewhite adipose tissue (WAT), which stores energy, and brown adiposetissue (BAT), which generates body heat.

Brown adipose tissue (BAT) is known to function in the dissipation ofchemical energy in response to cold or excess feeding, and also has thecapacity to modulate energy balance. Activation of brown adipose tissuehave been shown to improve glucose homeostasis and insulin sensitivityin humans suggesting that anyone with impaired insulin function mightbenefit from BAT activation (Stanford et al., J Clin Invest. 2013,123(1): 215-223).

Beige adipose tissues are generated as a result of browning of WAT, alsoknown as bciging. This occurs when adipocytes within WAT depots developfeatures of BAT. Beige adipocytes take on a multilocular appearance(containing several lipid droplets) and increase expression ofuncoupling protein 1 (UCP1). In doing so, these normally energy-storingwhite adipocytes become energy-releasing adipocytes (Harms et al. NatureMedicine. 2013, 19 (10): 1252-63).

Visceral fat or abdominal fat (also known as organ fat orintra-abdominal fat) is located inside the abdominal cavity, packedbetween the organs (stomach, liver, intestines, kidneys, etc.). Visceralfat is different from subcutaneous fat underneath the skin, andintramuscular fat interspersed in skeletal muscles. Fat in the lowerbody, as in thighs and buttocks, is subcutaneous and is not consistentlyspaced tissue, whereas fat in the abdomen is mostly visceral andsemi-fluid. An excess of visceral fat is known as central obesity, or“belly fat”, in which the abdomen protrudes excessively and newdevelopments such as the Body Volume Index (BVI) are specificallydesigned to measure abdominal volume and abdominal fat. Excess visceralfat is also linked to type 2 diabetes, insulin resistance, inflammatorydiseases and other obesity-related diseases (Mokdad et al., JAMA: TheJournal of the American Medical Association. 2001, 289 (1): 76-9).

Mass of adipose tissue can be determined by any method known to a personof ordinary skill in the art. For example, adipose tissue may bemeasured by dual-energy X-Ray absorptiometry (DXA), as demonstrated inExample 11 of the present application. Administration of the myostatininhibitor, e.g., antibody, or antigen binding fragment thereof, thatspecifically binds pro/latent myostatin increases the level of brownadipose tissue and/or the level of beige adipose tissue in the humansubject. On the other hand, administration of the myostatin inhibitor,e.g., anti-pro/latent myostatin antibody, or antigen-binding portionthereof, decreases the level of white adipose tissue and visceraladipose tissue in the human subject.

In some embodiments, the level of brown or beige adipose tissue isincreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In otherembodiments, the level of brown or beige adipose tissue is increased byat least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%,20-60%, 30-80%, 40-90%, or 50-100%.

In some embodiments, the level of white or visceral adipose tissue isdecreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In otherembodiments, the level of white or visceral adipose tissue is decreasedby at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%,20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

E. Effect on the Ratio of Adipose-to-Muscle Tissue in the Human Subject

Administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, that specifically binds pro/latent myostatindecreases the ratio between adipose-to-muscle tissue in the humansubject. In some embodiments, the ratio between adipose-to-muscle tissueis decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In otherembodiments, the ratio between adipose-to-muscle tissue is decreased byat least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%,20-60%, 30-80%, 40-90%, or 50-100%.

Administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, that specifically binds pro/latent myostatinalso increases the ratio of muscle tissue to adipose in the humansubject. In some embodiments, the ratio between muscle tissue-to-adiposeis increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In otherembodiments, the ratio between muscle tissue-to-adipose is increased byat least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%,20-60%, 30-80%, 40-90%, or 50-100%.

F. Effect on Glucose Uptake in the Human Subject

Administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, that specifically binds pro/latent myostatinaffects glucose uptake by tissues in the human subject. In someembodiments, glucose uptake by muscle tissue is increased. For example,glucose uptake by the muscle tissue is increased by at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,60%, 70%, 80%, 90% or 100%. In some embodiments, glucose uptake by themuscle tissue is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%,1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

In other embodiments, glucose uptake by white adipose tissue, livertissue and blood vessel tissue are reduced. In some embodiments, glucoseuptake by white adipose tissue, liver tissue and blood vessel tissue arereduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In otherembodiments, glucose uptake by white adipose tissue, liver tissue andblood vessel tissue are reduced by at least about 1-5%, 5-10%, 10-20%,1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

G. Effect on Muscle Catabolism of Protein and/or Muscle Release of AminoAcids in the Human Subject

Administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, that specifically binds pro/latent myostatindecreases muscle catabolism of protein and/or muscle release of aminoacids in the human subject. In some embodiments, muscle catabolism ofprotein and/or muscle release of amino acids is decreased by at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, musclecatabolism of protein and/or muscle release of amino acids is decreasedby at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%,20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

H. Effect on Insulin Dependent Glycemic Control in the Human Subject

Administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, that specifically binds pro/latent myostatinincreases insulin dependent glycemic control in the human subject. Insome embodiments, insulin dependent glycemic control is increased by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, insulindependent glycemic control is increased by at least about 1-5%, 5-10%,10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or50-100%.

I. Effect on Intramuscular Fat Infiltration in the Human Subject

Administration of the myostatin inhibitor, e.g., antibody, or antigenbinding fragment thereof, that specifically binds pro/latent myostatindecreases intramuscular fat infiltration in the human subject. In someembodiments, intramuscular fat infiltration is decreased by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, intramuscular fatinfiltration is decreased by at least about 1-5%, 5-10%, 10-20%, 1-30%,1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

J. Effect on Life Quality of the Human Subject

Assessment of the quality of life in patients with severe or chronicconditions, such as SCI patients, may involve integrated approaches toevaluate various aspects of physical, mental, social and otherparameters. Generally, a greater degree of quality of life is associatedwith factors such as: accessibility to assistive technology; communityreintegration; functionality with lower limb and walking and/or wheeledmobility; mental health; severity in neurological impairment andautonomic dysfunction; pain management; functional independence andself-care; upper limb strength; and spasticity control. Administrationof the antibody, or antigen binding fragment thereof, that specificallybinds pro/latent myostatin increases the quality of life of the humansubject to achieve a clinically meaningful improvement as measured by astandardized quality-of-life test/system. A number of suitable tests forassessing the quality of life in patients are known in the art,including: Incontinence Quality of Life Questionnaire (I-QOL); LifeSatisfaction Questionnaire (LISAT-9, LISAT-11); Quality of Life Index(QLI)—SCI Version; Quality of Life Profile for Adults with PhysicalDisabilities (QOLP-PD); Quality of Well Being (QWB) and Quality of WellBeing-Self-Administered (QWB-SA); Qualiveen; Satisfaction with LifeScale (SWLS, Deiner Scale); Short Form 36 (SF-36); Sickness ImpactProfile 68 (SIP 68); and World Health Organization Quality of Life-BREF(WHOQOL-BREF).

In some embodiments, quality of life is assessed in accordance with theSF-36 Quality of Life Scoring System, which is a validated scoringsystem, in which an 8-point change is considered clinically meaningful.Typically, for SCI patients, values are in the low 50's. In someembodiments, administration of an effective amount of the antibody, orantigen binding fragment thereof, that specifically binds pro/latentmyostatin results in a clinically meaningful improvement in astandardized quality-of-life test score. As used the herein, the term“clinically meaningful improvement” refers to a significant improvementover a standard level. In some embodiments, an SCI patient's SF-36Quality of Life scores are increased by at least 8 points, followingtreatment with an effective amount of an antibody or antigen-bindingfragments thereof described herein, as compared to the patient's scoreprior to the treatment. In some embodiments, patients achieve higherscores as assessed by the SF-36 Quality of Life Test, for example, atleast 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50points increase in the scores from the SF-36 Quality of Life ScoringSystem. In other embodiments, the scores from the SF-36 Quality of LifeScoring System is increased by at least about 8-10, 10-15, 15-20, 20-30,30-40, 40-50, 8-20, 8-30, 8-40, or 8-50.

In some embodiments, the SCI Neurological Quality of Life Test isemployed to assess patients' quality of life before and after treatmentwith the inhibitors of myostatin signaling disclosed herein. Advantagesof this test include: i) it is easy to administer; ii) it assesses bothphysical function and mental health; and, iii) it is highly validatedfor a number of clinical indications.

K. Effect on Preventing Muscle Loss or Atrophy in the Human Subject

Administration of an effective amount of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, that specifically bindspro/latent myostatin prevents muscle loss or atrophy in the humansubject at risk of developing muscle loss and/or atrophy. In someembodiments, muscle loss or atrophy is decreased or prevented by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%. In other embodiments, muscleloss or atrophy is decreased or prevented by at least about 1-5%, 5-10%,10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or50-100%.

In some embodiments, a suitable subject is a subject who has notdeveloped atrophy but is considered at risk of developing atrophy. Insome embodiments, a subject has a disease or condition associated with aneurological defect that impairs motor neuron function. In someembodiments, such conditions are caused by muscular dystrophy oratrophy. In some embodiments, the neurological defect is caused by anerve injury. In some embodiments, the nerve injury involves partialdenervation of motor neurons, which causes partial impairment offunction in the affected muscle. In some embodiments, such condition iscaused by SCI. In some embodiments, the subject with SCI is in an acuteor sub-acute phase of SCI (e.g., not yet reached a chronic phase).

In some embodiments, when a composition comprising an effective amountof an inhibitor of myostatin signaling described herein is administeredto a population of patients who are at risk of developing muscle atrophyassociated with partial denervation of motor neurons, the composition i)prevents manifestation or aggravation of the muscle atrophy in astatistically significant fraction of the patient population; or, ii)lessens the severity of the muscle atrophy in the statisticallysignificant fraction of the patient population.

Prevention of muscle loss or atrophy by the use of a myostatininhibitor, e.g., an antibody or antigen-binding fragment thereof,described herein can be readily monitored or assessed by any suitablemethods to evaluate motor function involving affected muscles.

In some embodiments, administration of an effective amount of suchantibody also prevents or lessens an early-onset axonal polyneuropathyin affected limbs.

L. Effect on Preventing Development of Metabolic Disease in the Subject

Administration of an effective amount of the myostatin inhibitor, e.g.,antibody, or antigen binding fragment thereof, that specifically bindspro/latent myostatin prevents development of metabolic disease in thesubject, e.g., a human subject. In some embodiments, development ofmetabolic disease is decreased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%or 100%. In other embodiments, development of metabolic disease isdecreased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%,10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

In some embodiments, a suitable subject is a subject who has not fullydeveloped a metabolic disease but is considered at risk of developingsuch a condition. In some embodiments, a subject has a disease orcondition associated with muscle dysfunction. In some embodiments, themuscle dysfunction is associated with partial denervation of motorneurons, which causes partial impairment of function in the affectedmuscle. In some embodiments, such conditions are caused by musculardystrophy or atrophy. In some embodiments, such condition is caused bySCI. In some embodiments, the subject with SCI is in an acute orsub-acute phase of SCI (e.g., not yet reached a chronic phase).

In some embodiments, when a composition comprising an effective amountof an inhibitor of myostatin signaling described herein is administeredto a population of patients who are at risk of developing a metabolicdisorder associated with muscle dysfunction, the composition i) preventsmanifestation or aggravation of the metabolic disorder in astatistically significant fraction of the patient population; or, ii)lessens the severity of the metabolic disease in the statisticallysignificant fraction of the patient population.

In some embodiments, effects on metabolism may be monitored or measuredby insulin resistance, lipid panel/markers (e.g., leptin), inflammatorymarkers and oxidative stress markers, including, but are not limited to:IL-6, TNF, CRP, plasma total antioxidant status, lipid oxidation anderythrocyte glutathione peroxidase activity.

Pharmaceutical Compositions

Myostatin inhibitors, e.g., antibodies, or antigen binding fragmentsthereof, described herein may be formulated into pharmaceuticalcompositions suitable for administration in human or non-human subjects.Such pharmaceutical compositions may be intended for therapeutic use, orprophylactic use. One or more of the myostatin inhibitors, e.g.,anti-pro/latent-myostatin antibodies can be mixed with apharmaceutically acceptable carrier (excipient), including buffer, toform a pharmaceutical composition for administering to a patient who maybenefit from reduced myostatin signaling in vivo. “Pharmaceuticallyacceptable” means that the carrier must be compatible with the activeingredient of the composition (and preferably, capable of stabilizingthe active ingredient) and not deleterious to the subject to be treated.Examples of pharmaceutically acceptable excipients (carriers), includingbuffers, would be apparent to the skilled artisan and have beendescribed previously. See, e.g., Remington: The Science and Practice ofPharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E.Hoover. Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations used, and may comprisebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Pharmaceutically acceptable excipients are further described herein.

In one example, a pharmaceutical composition described herein containsmore than one myostatin inhibitor, e.g., more than oneanti-pro/latent-myostatin antibody, or antigen-binding portion thereof,that recognize different epitopes/residues of the target antigen.

In some examples, the pharmaceutical composition described hereincomprises emulsion-based or lipid-based formulations, such as liposomescontaining a myostatin inhibitor, e.g., anti-pro/latent-myostatinantibody or antigen-binding portion thereof, which can be prepared byany suitable method, such as described in Epstein, et al., Proc. Natl.Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomeswith enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

The myostatin inhibitor, e.g., anti-pro/latent-myostatin antibody, orantigen-binding portion thereof, may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macrocmulsions. Exemplary techniques have beendescribed previously, sec, e.g., Remington, The Science and Practice ofPharmacy 20th Ed. Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein canbe formulated in sustained-release format. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, or antigen-binding portionthereof, which matrices are in the form of shaped articles, e.g. films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(v nylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), sucrose acetateisobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administrationmust be sterile. This is readily accomplished by, for example,filtration through sterile filtration membranes. Therapeutic antibodycompositions are generally placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosageforms such as tablets, pills, capsules, powders, granules, solutions orsuspensions, or suppositories, for oral, parenteral or rectaladministration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient can be mixed with a pharmaceutical carrier, e.g.,conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g., water, toform a solid preformulation composition containing a homogeneous mixtureof a compound of the present disclosure, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 mg to about 500 mg of the active ingredient of thepresent disclosure. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) andother sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g. egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%.

The emulsion compositions can be those prepared by mixing ananti-pro-myostatin antibody with Intralipid™ or the components thereof(soybean oil, egg phospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation includesolutions and suspensions in pharmaceutically acceptable, aqueous ororganic solvents, or mixtures thereof, and powders. The liquid or solidcompositions may contain suitable pharmaceutically acceptable excipientsas set out above. In some embodiments, the compositions are administeredby the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner

The Subject

Pharmaceutical compositions described herein are suitable foradministration in human or non-human subjects. Accordingly, themyostatin inhibitor, e.g., anti-pro/latent-myostatin antibodies, andantigen-binding portions thereof, described herein are useful asmedicament for administering to a subject who is likely to benefit fromreduced myostatin signaling. In some embodiments, suitable subjectsinclude healthy individuals who may nonetheless benefit from enhancedmuscle mass/function, as well as improved metabolism. In someembodiments, suitable subjects have an existing muscle condition and/orassociated metabolic dysfunction. In some embodiments, suitable subjectsare at risk of developing such condition(s). In some embodiments,suitable subjects are those on a therapy comprising another therapeuticagent to treat a muscle/metabolic condition, but which is associatedwith adverse effects or toxicity. In some embodiments, the subject is apediatric subject, e.g., human patients of between birth and <18 yearsof age.

In some embodiments, preferred subjects meet at least two of thefollowing criteria: i) the subject has a condition associated withpartial denervation of a motor neuron; ii) the condition involves amuscle containing or enriched with fast twitch fibers; and, iii) thesubject retains an anabolic capability (e.g., generally healthy adultswith injury) and/or is in a growth phase (e.g., young children, etc.).

In some embodiments, such medicament is suitable for administration in apediatric population, an adult population, and/or an elderly population.

The pediatric population in need for the myostatin inhibitor, e.g.,anti-pro/latent-myostatin antibodies and antigen-binding portionsthereof, described herein may range between 0 and 6 months of age,between 0 and 12 months of age, between 0 and 18 months of age, between0 and 24 months of age, between 0 and 36 months of age, between 0 and 72months of age, between 6 and 36 months of age, between 6 and 36 monthsof age, between 6 and 72 months of age, between 12 and 36 months of age,between 12 and 72 months of age. In some embodiments, the pediatricpopulation suitable for receiving the myostatin inhibitor, e.g.,antibody or antigen-binding fragment, described herein who is likely tobenefit from such treatment may range between 0 and 6 years of age,between 0 and 12 years of age, between 3 and 12 years of age, between 0and 17 years of age. In some embodiments, the population has an age ofat least 5 years, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17years. In some embodiments, the pediatric population may be aged below18 years old. In some embodiments, the pediatric population may be (a)at least 5 years of age and (b) below 18 years of age.

The adult population in need for the myostatin inhibitor, e.g.,anti-pro/latent-myostatin antibodies and antigen-binding portionsthereof, described herein may have an age of at least 18 years, e.g., atleast 19, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65 years. In someembodiments, the adult population may be below 65 years of age. In someembodiments, the adult population may of (a) at least 18 years of ageand (b) below 65 years of age.

The elderly population in need for the myostatin inhibitor, e.g.,anti-pro/latent-myostatin antibodies and antigen-binding portionsthereof, described herein may have an age of 65 years or older (i.e.,≥65 years old), e.g., at least 70, 75 or 80 years.

A human subject who is likely to benefit from the treatment may be ahuman patient having, at risk of developing, or suspected of having ametabolic disease/disorder associated with impaired neurologicalsignaling, such as those described below. A subject having apro/latent-myostatin-associated disease or disorder can be identified byroutine medical examination, e.g., laboratory tests, organ functionaltests, CT scans, or ultrasounds. A subject suspected of having any ofsuch disease/disorder might show one or more symptoms of thedisease/disorder. A subject at risk for the disease/disorder can be asubject having one or more of the risk factors for thatdisease/disorder.

A control subject, as described herein, is a subject who provides anappropriate reference for evaluating the effects of a particulartreatment or intervention of a test subject or subject. Control subjectscan be of similar age, race, gender, weight, height, and/or otherfeatures, or any combination thereof, to the test subjects.

In some embodiments, a myostatin assay (e.g., myostatin ELISA) is usedto determine a subject requiring treatment of an anti-pro/latentmyostatin antibody. Methods for assaying myostatin can be found inLakshman et al. Molecular and Cell Endocrinology (2009) 302:26-32(myostatin ELISA) and Bergen et al. Skeletal Muscle (2015) 5:21 (liquidchromatography with tandem mass spectrometry, both of which areincorporated by reference herein.

In some embodiments, methods are provided for improving muscleperformance in a subject. The subject may or may not have or be at riskof having a condition associated with decreased muscle mass and/ordecreased muscle function. As used herein, the term “muscle performance”generally refers to the capacity of the muscle to contract and/or toapply a force (e.g., to an external object). In some embodiments, muscleperformance may relate to the capacity of the muscle to consume energy.For example, in some embodiments, muscle performance may relate to thecapacity of the muscle to produce and/or consume adenosine triphosphate(ATP) molecules to facilitate muscle contraction. In some embodiments,muscle performance refers to the capacity of the muscle to contractrepeatedly for a particular duration of time. In some embodiments,muscle performance refers to the capacity of the muscle to apply a forceto an object, e.g., to move the object over a measurable distance. Insome embodiments, muscle performance refers to the capacity of themuscle to apply a force to an object for a particular duration of time(e.g., to move the object over a measurable distance for a particularduration of time).

In some embodiments, the myostatin inhibitor, e.g.,anti-pro/latent-myostatin antibody and antigen-binding portions thereof,described herein is administered to a subject in need of the treatmentat an amount sufficient to inhibit the proteolytic activation ofpro/latent-myostatin to active myostatin by at least 20% (e.g., 30%,40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo. In other embodiments,a myostatin inhibitor, e.g., antibody or antigen-binding portionthereof, is administered in an amount effective in reducing thepro/latent-myostatin or latent myostatin level by at least 20% (e.g.,30%, 40%, 50%, 60%, 70%, 80%, 90% or greater).

In some embodiments, the myostatin inhibitor, e.g.,anti-pro/latent-myostatin antibody or antigen-binding portion thereof,described herein is administered to a subject who will benefit fromincreased muscle mass. In some embodiments, the myostatin inhibitor,e.g., anti-pro/latent-myostatin antibody or antigen-binding portionthereof, described herein is administered to a subject who will benefitfrom increased muscle-to-fat ratios. In some embodiments, the myostatininhibitor, e.g., anti-pro/latent-myostatin antibody or antigen-bindingportion thereof, described herein is administered to a subject who willbenefit from increased muscle function. In some embodiments, the subjectmay or may not have or be at risk of having a condition associated withdecreased muscle mass and/or decreased muscle function. In someembodiments, the subject has or is at risk of having a conditionassociated with dccrcascd muscle mass and/or dccrcascd muscle function.

The methods of the present invention further comprising selecting asubject. In some embodiment, the subject suffer from or is at risk ofdeveloping a muscle condition or disorder. In some embodiment, thesubject suffer from or is at risk of developing a metabolic disorder. Insome embodiment, the subject suffer from or is at risk of developing adisease or disorder associated with impaired neurological signaling.

Routes of Administration

To practice the method disclosed herein, an effective amount of thepharmaceutical composition described above can be administered to asubject (e.g., a human) in need of the treatment via a suitable route,such as intravenous administration, e.g., as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerebrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, inhalation or topical routes. Commercially availablenebulizers for liquid formulations, including jet nebulizers andultrasonic nebulizers are useful for administration. Liquid formulationscan be directly nebulized and lyophilized powder can be nebulized afterreconstitution. Alternatively, anti-pro/latent-myostatin antibodies canbe aerosolized using a fluorocarbon formulation and a metered doseinhaler, or inhaled as a lyophilized and milled powder.

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the pharmaceutical composition tothe subject, depending upon the type of disease to be treated or thesite of the disease. This composition can also be administered via otherconventional routes, e.g., administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional, and intracranial injection or infusion techniques. Inaddition, it can be administered to the subject via injectable depotroutes of administration such as using 1-, 3-, or 6-month depotinjectable or biodegradable materials and methods.

Injectable compositions may contain various carriers such as vegetableoils, dimethylactamide, dimethyformamide, ethyl lactate, ethylcarbonate, isopropyl myristate, ethanol, and polyols (glycerol,propylene glycol, liquid polyethylene glycol, and the like). Forintravenous injection, water soluble antibodies can be administered bythe drip method, whereby a pharmaceutical formulation containing theantibody and a physiologicallyacceptable excipients is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the antibody, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution.

In one embodiment, a myostatin inhibitor, e.g.,anti-pro/latent-myostatin antibody or antigen-binding portion thereof,is administered via site-specific or targeted local delivery techniques.Examples of site-specific or targeted local delivery techniques includevarious implantable depot sources of the myostatin inhibitor, e.g.,anti-pro/latent-myostatin antibody or antigen-binding portion thereof,or local delivery catheters, such as infusion catheters, an indwellingcatheter, or a needle catheter, synthetic grafts, adventitial wraps,shunts and stents or other implantable devices, site specific carriers,direct injection, or direct application. See, e.g., PCT Publication No.WO 00/53211 and U.S. Pat. No. 5,981,568.

The particular dosage regimen, e.g., dose, timing and repetition, usedin the method described herein will depend on the particular subject andthat subject's medical history.

Treatment efficacy for a disease/disorder associated with myopathy canbe assessed using any suitable methods. For example, treatment efficacyfor a disease/disorder associated with myopathy can be assessed byevaluating muscle weakness (e.g., assessing the pattern and severity ofweakness), electromyography, evaluating blood chemistries (e.g.,assessing electrolytes, assessing endocrine causes, measuring creatininekinase level, determining erythrocyte sedimentation rate and performingantinuclear antibody assays), and evaluating biopsies (e.g., byhistologic, histochemical, electron microscopic, biochemical, andgenetic analysis).

“An effective amount” as used herein refers to the amount of each activeagent required to confer a therapeutic effect on the subject, eitheralone or in combination with one or more other active agents. Forexample, an effective amount refers to the amount of a myostatininhibitor, e.g., an antibody, or antigen binding fragment thereof, ofthe present disclosure which is sufficient to achieve a biologicaleffect, e.g., an increase in muscle mass or muscle fiber diameter, aswitch in muscle fiber type, an increase in the amount of forcegenerated by the muscle, an increase in mass and/or function of a muscletissue in the subject; an increase in the metabolic rate of the subject;an increase in insulin sensitivity of the subject; an increase in alevel of brown adipose tissue in the subject; an increase in a level ofbeige adipose tissue in the subject; a decrease in a level of whiteadipose tissue in the subject; a decrease in a level of visceral adiposetissue in the subject; a decrease in ratio of adipose-to-muscle tissuein the subject; an increase in glucose uptake by a brown adipose tissue,a beige adipose tissue, or a muscle tissue in the subject; a decrease inglucose uptake by a white adipose tissue or a liver tissue; a decreasein muscle catabolism of protein and/or muscle release of amino acids inthe subject; an increase in insulin dependent glycemic control in thesubject; or a decrease in intramuscular fat infiltration in the subject;or a clinically significant outcome, e.g., a partial or completerecovery of the ability to perform physical tasks after injury; aclinically meaningful improvement in quality of life as assessed by astandardized system, such as SF-36 Quality of Life Scoring System;prevention of muscle loss or atrophy in the subject; and/or preventionof developing a metabolic disease in the subject.

Effective amounts vary, as recognized by those skilled in the art,depending on the particular condition being treated, the severity of thecondition, the individual patient parameters including age, physicalcondition, size, gender and weight, the duration of the treatment, thenature of concurrent therapy (if any), the specific route ofadministration and like factors within the knowledge and expertise ofthe health practitioner. These factors are well known to those ofordinary skill in the art and can be addressed with no more than routineexperimentation. It is generally preferred that a maximum dose of theindividual components or combinations thereof be used, that is, thehighest safe dose according to sound medical judgment. It will beunderstood by those of ordinary skill in the art, however, that apatient may insist upon a lower dose or tolerable dose for medicalreasons, psychological reasons or for virtually any other reasons.

In some embodiments, in the context of an increase in the level ofpro-myostatin in the target muscle, the increase is at least 1-fold,1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, or 10-fold or more (or any range bracketed byany of the values), compared to a control level of pro-myostatin. In oneembodiment, the increase in the level of pro-myostatin in the targetmuscle is an increase in a range of 1-fold to 3-fold, 1.2-fold to10-fold, 2-fold to 9-fold, 3-fold to 8-fold, 4-fold to 7-fold, 2-fold to7-fold, etc. compared to the control level of pro-myostatin.

In some embodiments, in the context of an increase in latent myostatinin the target muscle after the administering step, the increase isdetectable within 4 hours, 24 hours, 48 hours, 7 days, 14 days, 21 days,28 days or 30 days (or any time range bracketed by any of the listedduration of times) after the administering step. In one embodiment, anincrease in latent myostatin in the target muscle after theadministering step is detectable for at least 5 days, 7 days, 14 days,21 days, 28 days, or 30 days (or any time range bracketed by any of thelisted duration of times) after the administering step. In oneembodiment, an increase in the level of latent myostatin in the targetmuscle after the administering step is at least 1-fold, 1.2-fold,1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, or 10-fold or more (or any range bracketed by any of thevalues), compared to the level of latent myostatin in the target musclebefore the administering step. In one embodiment, an increase in thelevel of latent myostatin in the target muscle after the administeringstep is an increase in a range of 1-fold to 3-fold, 1.2-fold to 10-fold,2-fold to 9-fold, 3-fold to 8-fold, 4-fold to 7-fold, 2-fold to 7-fold,etc., compared to the level of latent myostatin in the target musclebefore the administering step.

In some embodiment, in the context of an increase in latent myostatin inthe circulation after the administering step, an increase is detectablewithin 4 hours, 24 hours, 48 hours, 7 days, 14 days, 21 days, 28 days,or 30 days (or any time range bracketed by any of the listed duration oftimes) after the administering step. In one embodiment, an increase inlatent myostatin in the circulation after the administering step isdetectable for at least 5 days, 7 days, 14 days, 21 days, 28 days, or 30days (or any time range bracketed by any of the listed duration oftimes) after the administering step. In one embodiment, an increase inthe level of latent myostatin in the circulation after the administeringstep is at least 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 15-fold,20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold or more(or any range bracketed by any of the values), compared to the level oflatent myostatin in the circulation before the administering step. Inone embodiment, an increase in the level of latent myostatin in thetarget muscle after the administering step is an increase in a range of1-fold to 3-fold, 1.2-fold to 10-fold, 2-fold to 9-fold, 3-fold to8-fold, 4-fold to 7-fold, 2-fold to 7-fold, etc., compared to the levelof latent myostatin in the target muscle before the administering step.

In some embodiments, in the context of a decrease in the level of latentmyostatin in the circulation, the decrease is at least 1-fold, 1.2-fold,1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, or 10-fold or more (or any range bracketed by any of thevalues), compared to a control level of latent myostatin. In oneembodiment, a decrease in the level of latent myostatin in thecirculation is a decrease in a range of 1-fold to 3-fold, 1.2-fold to10-fold, 2-fold to 9-fold, 3-fold to 8-fold, 4-fold to 7-fold, 2-fold to7-fold, etc. compared to the control level of latent myostatin.

As discussed above, in some embodiments, in the context ofadministration of a myostatin inhibitor, e.g., a pro/latent-myostatinantibody, or antigen binding fragment thereof, to a subject, aneffective amount is an amount effective to increase mass of a targetmuscle in the subject compared with a control muscle mass. In someembodiments, muscle treated with an effective amount of the antibody isincrease by at least 1%, at least 2%, at least 3%, at least 4%, at least5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, atleast 11%, at least 12%, at least 13%, at least 14%, at least 15%, atleast 16%, at least 17%, at least 18%, at least 19%, at least 20%, etc.as compared with a control muscle mass that is not treated with aneffective amount of the antibody. In some embodiments, such muscle massincrease is achieved in a select group or type of muscles in thesubject.

In some embodiments, in the context of administration of a myostatininhibitor, e.g., pro/latent-myostatin antibody, or antigen bindingfragment thereof, to a subject, an effective amount is an amounteffective to switch fiber types in the subject. In some embodiments, aneffective amount of the antibody can promote a fiber type switch fromtype I to type II. In some embodiments, an effective amount of themyostatin inhibitor, e.g., antibody or antigen-binding portion thereof,can promote a fiber type switch from type I to type IIB. In someembodiments, an effective amount of the myostatin inhibitor, e.g.,antibody or antigen-binding portion thereof, can promote type II fibers,relative to other types of fibers. In some embodiments, an effectiveamount of the myostatin inhibitor, e.g., antibody or antigen-bindingportion thereof, can promote type JIB fibers, relative to other types offibers. In some embodiments, such phenotypic switch in fibers may occurwithout significant change in overall muscle mass. In other embodiments,such phenotypic switch in fibers may coincide an increase in overallmuscle mass.

In some embodiments, in the context of administration of a myostatininhibitor, e.g., pro/latent-myostatin antibody, or antigen bindingfragment thereof, to a subject, an effective amount is an amounteffective to increase diameter of muscle fiber in the subject comparedwith a control muscle fiber. In some embodiments, the increase in thediameter of the muscle fiber is an increase of at least 1.1-fold, atleast 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold,at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least1.9-fold, at least 2-fold, at least 4-fold, at least 5-fold or morecompared with a control muscle fiber. In some embodiments, the increasein the diameter of muscle fiber is an increase in a range of 1-fold to5-fold, 2-fold to 10-fold, 1-fold to 1.5-fold, 1-fold to 2-fold, etc.compared with a control muscle fiber.

In some embodiments, in the context of administration of a myostatininhibitor, e.g., pro/latent-myostatin antibody, or antigen bindingfragment thereof, to a subject, an effective amount is an amounteffective to increase muscle-to-fat ratio in the subject compared with acontrol muscle mass. In some embodiments, the increase in themuscle-to-fat ratio is an increase of at least 1.1-fold, at least1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, atleast 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold,at least 2-fold, at least 4-fold, at least 5-fold or more compared witha control subject. In some embodiments, the increase in themuscle-to-fat ratio is an increase in a range of 1-fold to 5-fold,2-fold to 10-fold, 1-fold to 1.5-fold, 1-fold to 2-fold, etc. comparedwith a control subject.

In some embodiments, in the context of administration of a myostatininhibitor, e.g., a pro/latent-myostatin antibody, or antigen bindingfragment thereof, to a subject, an effective amount is an amounteffective to decrease intramuscular fat infiltration in the subjectcompared with a control muscle mass. In some embodiments, the decreasein the intramuscular fat infiltration is a decrease of at least1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, atleast 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold,at least 1.9-fold, at least 2-fold, at least 4-fold, at least 5-fold ormore compared with a control subject. In some embodiments, the decreasein intramuscular fat infiltration is a decrease in a range of 1-fold to5-fold, 2-fold to 10-fold, 1-fold to 1.5-fold, 1-fold to 2-fold, etc.compared with a control subject.

In some embodiments, a method of preventing a reduction of and/orincreasing muscle mass in a human subject includes administering amyostatin inhibitor, e.g., a pro/latent-myostatin antibody, or antigenbinding fragment thereof, to a subject that inhibits proteolyticformation of mature myostatin by a tolloid protease. In one embodiment,inhibition of proteolytic cleavage of pro-myostatin or latent myostatinby a tolloid protease results in a progressive increase in muscle mass.In one embodiment, a subject exhibits a progressive increase in musclemass for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12weeks, 14 weeks, 16 weeks, 18 weeks, or 20 weeks (or any range bracketedby any of the values). In some embodiments, a method of preventing areduction of and/or increasing muscle mass in a human subject includesadministering a myostatin inhibitor, e.g., a pro/latent-myostatinantibody, or antigen binding fragment thereof, to a subject comprisingmore than two doses. In one embodiment, administering a myostatininhibitor, e.g., a pro/latent-myostatin antibody, or antigen bindingfragment thereof, comprises at least a first dose and a second dose, thefirst dose and the second dose are administered to the subject at leastabout 2 weeks apart, 4 weeks apart, 6 weeks apart, 8 weeks apart, or 12weeks apart.

In some embodiments, in the context of administration of a myostatininhibitor, e.g., a pro/latent-myostatin antibody, or antigen bindingfragment thereof, to a subject, an effective amount is an amounteffective to increase function of a target muscle in the subjectcompared with a control muscle function. In some embodiments, theincrease in muscle function is an increase of at least 1.1-fold, atleast 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold,at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least1.9-fold, at least 2-fold, at least 4-fold, at least 5-fold or morecompared with a control muscle function. In some embodiments, theincrease in muscle function is an increase in a range of 1-fold to5-fold, 2-fold to 10-fold, 1-fold to 1.5-fold, 1-fold to 2-fold, etc.compared with a control muscle function.

As used herein, the term “control muscle mass” refers to a referencestandard useful for evaluating effects of a condition (e.g., treatmentwith a myostatin inhibitor, e.g., a pro/latent-myostatin antibody, orantigen binding fragment thereof) on the mass of a target muscle in asubject. In some embodiments, a control muscle mass is a predeterminedvalue. In some embodiments, a control muscle mass is experimentallydetermined. In some embodiments, a control muscle mass is the mass of atarget muscle in a subject who has not been administered the myostatininhibitor, e.g., pro/latent-myostatin antibody, or antigen bindingfragment thereof. In some embodiments, a control muscle mass is the mass(e.g., the average mass) of a target muscle in a population of subjectswho have not been administered the myostatin inhibitor, e.g.,pro/latent-myostatin antibody, or antigen binding fragment thereof. Insome embodiments, a control muscle mass is the mass of a target musclein a subject prior to (e.g., immediately prior to) being administeredthe myostatin inhibitor, e.g., pro/latent-myostatin antibody, or antigenbinding fragment thereof. In some embodiments, a control muscle mass isthe mass of a target muscle in a subject who has been administered, inplace of the myostatin inhibitor, e.g., pro/latent-Myostatin antibody,or antigen binding fragment thereof, a normal antibody (e.g., of thesame isotype as the pro/latent-Myostatin antibody) that has beenobtained from an animal that has not been exposed to the antigen towhich the pro/latent-myostatin antibody, or antigen binding fragmentthereof, is directed. In some embodiments, a control muscle mass is themass of a target muscle in a subject who has been administered, in placeof the myostatin inhibitor, e.g., pro/latent-myostatin antibody, orantigen binding fragment thereof, a vehicle, e.g., saline.

In some embodiments, in the context of administration of a myostatininhibitor, e.g., pro/latent-myostatin antibody, or antigen bindingfragment thereof, to a subject, an effective amount is an amounteffective to increase force generation capacity (e.g., a maximal forcegeneration as determined in vitro with a muscle lever system adaptedwith a horizontal perfusion bath) of a target muscle in the subjectcompared with a control force generation capacity. In some embodiments,the increase in force generation capacity is an increase of at least1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, atleast 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold,at least 1.9-fold, at least 2-fold, at least 4-fold, at least 5-fold ormore compared with a control force generation capacity. In someembodiments, the increase in force generation capacity is an increase ina range of 1-fold to 5-fold, 2-fold to 10-fold, 1-fold to 1.5-fold,1-fold to 2-fold, etc. compared with a control force generationcapacity.

As used herein, the term “control force generation capacity” refers to areference standard useful for evaluating effects of a condition (e.g.,treatment with a pro/latent-myostatin antibody, or antigen bindingfragment thereof) on the force generation capacity of a muscle in asubject. In some embodiments, a control force generation capacity is apredetermined value. In some embodiments, a control force generationcapacity is experimentally determined. In some embodiments, a controlforce generation capacity is the force generation capacity of a targetmuscle in a subject who has not been administered the myostatininhibitor, e.g., pro/latent-myostatin antibody, or antigen bindingfragment thereof. In some embodiments, a control force generationcapacity is the force generation capacity (e.g., the average forcegeneration capacity) of a target muscle in a population of subjects whohave not been administered the myostatin inhibitor, e.g.,pro/latent-myostatin antibody, or antigen binding fragment thereof. Insome embodiments, a control force generation capacity is the forcegeneration capacity of a target muscle in a subject prior to (e g,immediately prior to) being administered the myostatin inhibitor, e.g.,pro/latent-myostatin antibody, or antigen binding fragment thereof. Insome embodiments, a control force generation capacity is the forcegeneration capacity of a target muscle in a subject who has beenadministered, in place of the myostatin inhibitor, e.g.,pro/latent-myostatin antibody, a normal antibody (e.g., of the sameisotype as the pro/latent-myostatin antibody) that has been obtainedfrom an animal that has not been exposed to the antigen to which thepro/latent-myostatin antibody is directed. In some embodiments, acontrol force generation capacity is the force generation capacity of atarget muscle in a subject who has been administered, in place of themyostatin inhibitor, e.g., pro/latent-myostatin antibody, or antigenbinding fragment thereof, a vehicle, e.g., saline.

In some embodiments, the target muscle is a plantarflexor muscle. Insome embodiments, the target muscle is a muscle containing type 2fibers. In some embodiments, the target muscle is a muscle containingfast oxidative fibers or fast glycolytic fibers. In some embodiments,the target muscle is a muscle containing type IIB fibers. In someembodiments, the administration of myostatin inhibitor, e.g.,pro/latent-myostatin antibody, or antigen binding fragment thereof,results in increase in type IIB fiber cross-sectional area by at least1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% (or any rangebracketed by any of the values), compared to the cross-sectional areabefore the administering step.

Dosages

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, antibodiesand antigen-binding portions thereof that are compatible with the humanimmune system, such as humanized antibodies or fully human antibodies,may be uscd to prolong half-life of the antibody and to prevent theantibody being attacked by the host's immune system. Frequency ofadministration may be determined and adjusted over the course oftherapy, and is generally, but not necessarily, based on treatmentand/or suppression and/or amelioration and/or delay of adisease/disorder associated with myopathy. Alternatively, sustainedcontinuous release formulations of a myostatin inhibitor, e.g., ananti-pro/latent-myostatin antibody, or antigen-binding portion thereof,may be appropriate. Various formulations and devices for achievingsustained release would be apparent to the skilled artisan and arewithin the scope of this disclosure.

In one example, dosages for a myostatin inhibitor, e.g., ananti-pro/latent-myostatin antibody, or antigen binding fragment thereof,as described herein may be determined empirically in individuals whohave been given one or more administration(s) of the myostatininhibitor, e.g., antibody, or antigen binding fragment thereof.Individuals are given incremental dosages of the antagonist. To assessefficacy of the antagonist, an indicator of the disease/disorder can befollowed.

Generally, for administration of any of the antibody, or antigen bindingfragment thereof, described herein, an initial candidate dosage can beabout 2 mg/kg. For the purpose of the present disclosure, a typicaldaily dosage might range from about any of 0.1 pg/kg to 3 μg/kg to 30pg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of symptoms occurs or untilsufficient therapeutic levels are achieved to alleviate a disease ordisorder associated with pro/latent-myostatin, or a symptom thereof. Anexemplary dosing regimen comprises administering an initial dose ofabout 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg ofthe antibody, or antigen binding fragment thereof, or followed by amaintenance dose of about 1 mg/kg every other week. However, otherdosage regimens may be useful, depending on the pattern ofpharmacokinetic decay that the practitioner wishes to achieve. Forexample, dosing from one-four times a week is contemplated. In someembodiments, dosing ranging from about 3 μg/mg to about 2 mg/kg (such asabout 3 μg/mg, about 10 pg/mg, about 30 pg/mg, about 100 pg/mg, about300 μg/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In someembodiments, dosing frequency is once every week, every 2 weeks, every 4weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every9 weeks, or every 10 weeks; or once every month, every 2 months, orevery 3 months, every 4 months, every 5 months, every 6 months, every 8months, every 10 months, every year, or longer. The progress of thistherapy is easily monitored by conventional techniques and assays. Thedosing regimen (including the antibody used) can vary over time.

In some embodiments, the administration of any of the myostatininhibitors, e.g., antibodies, or antigen binding fragments thereof,described herein comprises a single dose. In some embodiments, theadministration of any of the myostatin inhibitors, e.g., antibodies, orantigen binding fragments thereof, described herein comprises multipledoses (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses).Administering may comprise more than two doses. In some embodiments, theadministration comprises at least a first dose and a second dose of atherapeutically effective amount of the myostatin inhibitor, e.g.,antibody or antigen-binding portion thereof. In one embodiment, thefirst dose and the second dose are administered to the subject at leastabout 4 weeks apart, 6 weeks apart, 8 weeks apart, or 12 weeks apart.

In some embodiments, for an adult patient of normal weight, dosesranging from about 0.3 to 5.00 mg/kg may be administered. The particulardosage regimen, e.g., dose, timing and repetition, will depend on theparticular individual and that individual's medical history, as well asthe properties of the individual agents (such as the half-life of theagent, and other relevant considerations).

For the purpose of the present disclosure, the appropriate dosage of amyostatin inhibitor, e.g., an anti-pro/latent-myostatin antibody, orantigen binding fragment thereof, will depend on the specific antibody(or compositions thereof) employed, the type and severity of thedisease/disorder, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antagonist, and the discretion of the attendingphysician. In some embodiments, a clinician will administer a myostatininhibitor, e.g., an anti-pro/latent-myostatin antibody orantigen-binding portion thereof, until a dosage is reached that achievesthe desired result. Administration of a myostatin inhibitor, e.g., ananti-pro/latent-myostatin antibody or antigen-binding portion thereof,can be continuous or intermittent, depending, for example, upon therecipient's physiological condition, whether the purpose of theadministration is therapeutic or prophylactic, and other factors knownto skilled practitioners. The administration of a myostatin inhibitor,e.g., an anti-pro/latent-myostatin antibody, or antigen binding fragmentthereof, may be essentially continuous over a preselected period of timeor may be in a series of spaced dose, e.g., either before, during, orafter developing a disease or disorder associated withpro/latent-myostatin.

As used herein, the term “treating” refers to the application oradministration of a composition including one or more active agents to asubject, who has a disease/disorder associated with myopathy, a symptomof the disease/disorder, or a predisposition toward thedisease/disorder, with the purpose to cure, heal, alleviate, relieve,alter, remedy, ameliorate, improve, or affect the disorder, the symptomof the disease, or the predisposition toward the disease/disorder.

Alleviating a disease/disorder associated with pro/latent-myostatinincludes delaying the development or progression of the disease, orreducing disease severity. Alleviating the disease does not necessarilyrequire curative results. As used therein, “delaying” the development ofa disease/disorder associated with pro/latent-myostatin means to defer,hinder, slow, retard, stabilize, and/or postpone progression of thedisease. This delay can be of varying lengths of time, depending on thehistory of the disease and/or individuals being treated. A method that“delays” or alleviates the development of a disease, or delays the onsetof the disease, is a method that reduces probability of developing oneor more symptoms of the disease in a given time frame and/or reducesextent of the symptoms in a given time frame, when compared to not usingthe method. Such comparisons are typically based on clinical studies,using a number of subjects sufficient to give a statisticallysignificant result.

Combination Therapies

The invention encompasses pharmaceutical compositions and relatedmethods used as combination therapies for treating subjects who maybenefit from myostatin inhibition in vivo. In any of these embodiments,such subjects may receive combination therapies that include a firstcomposition comprising at least one myostatin inhibitor, e.g., antibodyor antigen-binding portion thereof, described herein, in conjunctionwith a second composition comprising at least one additional therapeuticintended to treat the same or overlapping disease or clinical condition.The first and second compositions may both act on the same cellulartarget, or discrete cellular targets. In some embodiments, the first andsecond compositions may treat or alleviate the same or overlapping setof symptoms or aspects of a disease or clinical condition. In someembodiments, the first and second compositions may treat or alleviate aseparate set of symptoms or aspects of a disease or clinical condition.To give but one example, the first composition may treat myopathyassociated with a disease, while the second composition may treatinflammation or fibrosis associated with the same disease, etc. Suchcombination therapies may be administered in conjunction with eachother. The phrase “in conjunction with,” in the context of combinationtherapies, means that therapeutic effects of a first therapy overlapstemporarily and/or spatially with therapeutic effects of a secondtherapy in the subject receiving the combination therapy. Thus, thecombination therapies may be formulated as a single formulation forconcurrent administration, or as separate formulations, for sequentialadministration of the therapies.

In preferred embodiments, combination therapies produce synergisticeffects in the treatment of a disease. The term “synergistic” refers toeffects that are greater than additive effects (e.g., greater efficacy)of each monotherapy in aggregate.

In some embodiments, combination therapies comprising a pharmaceuticalcomposition described herein produce efficacy that is overall equivalentto that produced by another therapy (such as monotherapy of a secondagent) but are associated with fewer unwanted adverse effect or lesssevere toxicity associated with the second agent, as compared to themonotherapy of the second agent. In some embodiments, such combinationtherapies allow lower dosage of the second agent but maintain overallefficacy. Such combination therapies may be particularly suitable forpatient populations where a long-term treatment is warranted and/orinvolving pediatric patients.

Accordingly, the invention provides pharmaceutical compositions andmethods for use in combination therapies for the enhancement of musclemass/function and for the treatment or prevention of metabolic diseasesor diseases associated with an impaired neurological signaling,including diabetes, obesity and spinal cord injury. Accordingly, themethods or the pharmaceutical compositions further comprise a secondtherapy. In some embodiments, the second therapy may be useful intreating or preventing metabolic diseases or diseases associated with animpaired neurological signaling. The second therapy may diminish ortreat at least one symptom(s) associated with the targeted disease. Thefirst and second therapies may exert their biological effects by similaror unrelated mechanisms of action; or either one or both of the firstand second therapies may exert their biological effects by amultiplicity of mechanisms of action.

It should be understood that the pharmaceutical compositions describedherein may have the first and second therapies in the samepharmaceutically acceptable carrier or in a different pharmaceuticallyacceptable carrier for each described embodiment. It further should beunderstood that the first and second therapies may be administeredsimultaneously or sequentially within described embodiments.

The one or more anti-myostatin antibodies or other myostatin inhibitorsof the invention may be used in combination with one or more ofadditional therapeutic agents. Examples of the additional therapeuticagents which can be used with an anti-myostatin antibody of theinvention include, but are not limited to, diabetes mellitus-treatingagents, diabetic complication-treating agents, cardiovasculardiseases-treating agents, anti-hyperlipemic agents, hypotensive orantihypertensive agents, anti-obesity agents, nonalcoholicsteatohepatitis (NASH)-treating agents, chemotherapeutic agents,immunotherapeutic agents, immunosuppressive agents, and the like. Suchcombination therapies may advantageously utilize lower dosages of theadministered therapeutic agents, thus avoiding possible toxicities orcomplications associated with the various monotherapies. Examples ofagents for treating diabetes mellitus include insulin formulations(e.g., animal insulin formulations extracted from a pancreas of a cattleor a swine; a human insulin formulation synthesized by a geneengineering technology using microorganisms or methods), insulinsensitivity enhancing agents, pharmaceutically acceptable salts,hydrates, or solvates thereof (e.g., pioglitazone, troglitazone,rosiglitazone, netoglitazone, balaglitazone, rivoglitazone,tesaglitazar, farglitazar, CLX-0921, R-483, NIP-221, NIP-223, DRF-2189,GW-7282TAK-559, T-131, RG-12525, LY-510929, LY-519818, BMS-298585,DRF-2725, GW-1536, GI-262570, KRP-297, TZD18 (Merck), DRF-2655, and thelike), alpha-glycosidase inhibitors (e.g., voglibose, acarbose,miglitol, emiglitate and the like), higuanides (e.g., phenformin,metformin, buformin and the like) or sulfonylureas (e.g., tolbutamide,glibenclamide, gliclazide, chlorpropamide, tolazamide, acetohexamide,glyclopyramide, glimepiride and the like) as well as other insulinsecretion-promoting agents (e.g., repaglinide, senaglinide, nateglinide,mitiglinidc, GLP-1 and the like), amyrin agonist (e.g., pramlintidc andthe like), phosphotyrosin phosphatase inhibitor (e.g., vanadic acid andthe like) and the like.

Examples of agents for treating diabetic complications include, but arenot limited to, aldose reductase inhibitors (e.g., tolrestat,epalrestat, zenarestat, zopolrestat, minalrestat, fidareatat, SK-860,CT-112 and the like), neurotrophic factors (e.g., NGF, NT-3, BDNF andthe like), PKC inhibitors (e.g., LY-333531 and the like), advancedglycation end-product (AGE) inhibitors (e.g., ALT946, pimagedine,pyradoxamine, phenacylthiazolium bromide (ALT766) and the like), activeoxygen quenching agents (e.g., thioctic acid or derivative thereof, abioflavonoid including flavones, isoflavones, flavonones, procyanidins,anthocyanidins, pycnogenol, lutein, lycopene, vitamins E, coenzymes Q,and the like), cerebrovascular dilating agents (e.g., tiapride,mexiletene and the like).

In one embodiment, remission of diabetes can be induced byadministration of a myostatin inhibitor in combination with a caloricrestriction diet, or other diet.

Anti-hyperlipemic agents include, for example, statin-based compoundswhich are cholesterol synthesis inhibitors (e.g., pravastatin,simvastatin, lovastatin, atorvastatin, fluvastatin, rosuvastatin and thelike), squalene synthetase inhibitors or fibrate compounds having atriglyceride-lowering effect (e.g., fenofibrate, gemfibrozil,bezafibrate, clofibrate, sinfibrate, clinofibrate and the like), niacin,PCSK9 inhibitors, triglyceride lowing agents or cholesterol sequestingagents.

Hypotensive agents include, for example, angiotensin converting enzymeinhibitors (e.g., captopril, enalapril, delapril, benazepril,cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril,perindopril, quinapril, ramipril, trandolapril and the like) orangiotensin II antagonists (e.g., losartan, candesartan cilexetil,olmesartan medoxomil, eprosartan, valsartan, telmisartan, irbesartan,tasosartan, pomisartan, ripisartan forasartan, and the like) or calciumchannel blockers (e.g., amlodipine) or aspirin.

Nonalcoholic steatohepatitis (NASH)-treating agents include, forexample, ursodiol, pioglitazone, orlistat, betaine, rosiglitazone. Inone embodiment, steatosis, resulting liver inflammation, and fibrosis inNAFLD and/or NASH subjects can be treated by administration of amyostatin inhibitor in combination with a caloric restriction diet, orother diet.

Anti-obesity agents include, for example, central antiobesity agents(e.g., dexfenfluramine, fenfluramine, phentermine, sibutramine,amfepramone, dexamphetamine, mazindol, phenylpropanolamine, clobenzorexand the like), gastrointestinal lipase inhibitors (e.g., orlistat andthe like), beta 3-adrenoceptor agonists (e.g., CL-316243, SR-58611-A,UL-TG-307, SB-226552, AJ-9677, BMS-196085 and the like), peptide-basedappetite-suppressing agents (e.g., leptin, CNTF and the like),cholecystokinin agonists (e.g., lintitript, FPL-15849 and the like) andthe like.

Chemotherapeutic agents include, for example, alkyl ating agents (e.g.,cyclophosphamide, iphosphamide and the like), metabolism antagonists(e.g., methotrexate, 5-fluorouracil and the like), anticancerantibiotics (e.g., mitomycin, adriamycin and the like),vegetable-derived anticancer agents (e.g., vincristine, vindesine, taxoland the like), cisplatin, carboplatin, etoposide and the like. Amongthese substances, 5-fluorouracil derivatives such as furtulon andncofurtulon are preferred.

Immunotherapeutic agents include, for example, microorganisms orbacterial components (e.g., muramyl dipeptide derivative, picibanil andthe like), polysaccharides having immune potentiating activity (e.g.,lentinan, sizofilan, krestin and the like), cytokines obtained by a geneengineering technology (e.g., interferon, interleukin (IL) and thelike), colony stimulating factors (e.g., granulocyte colony stimulatingfactor, crythropoctin and the like) and the like, among thesesubstances, those preferred are IL-1, IL-2, IL-12 and the like.

Immunosuppressive agents include, for example, calcineurininhibitor/immunophilin modulators such as cyclosporine (Sandimmune,Gengraf, Neoral), tacrolimus (Prograf, FK506), ASM 981, sirolimus (RAPA,rapamycin, Rapamune), or its derivative SDZ-RAD, glucocorticoids(prednisone, prednisolone, methylprednisolone, dexamethasone and thelike), purine synthesis inhibitors (mycophenolate mofetil, MMF,CellCept®, azathioprine, cyclophosphamide), interleukin antagonists(basiliximab, daclizumab, deoxyspergualin), lymphocyte-depleting agentssuch as antithymocyte globulin (Thymoglobulin, Lymphoglobuline),anti-CD3 antibody (OKT3), and the like.

In addition, agents whose cachexia improving effect has been establishedin an animal model or at a clinical stage, such as cyclooxygenaseinhibitors (e.g., indomethacin and the like), progesterone derivatives(e.g., megestrol acetate), glucosteroid (e.g., dexamethasone and thelike), metoclopramide-based agents, tetrahydrocannabinol-based agents,lipid metabolism improving agents (e.g., eicosapentanoic acid and thelike), growth hormones, IGF-1, antibodies against TNF-α, LIF, IL-6 andoncostatin M may also be employed concomitantly with an anti-myostatinantibody according to the present invention. Additional therapeuticagents for use in the treatment of diseases or conditions related tometabolic disorders and/or impaired neurological signaling would beapparent to the skilled artisan and are within the scope of thisdisclosure.

In some embodiments, second agents suitable for administration as acombination therapy in conjunction with the antibodies described hereinare anti-fibrotic agents, such as TGF131 inhibitors.

In some embodiments, second agents suitable for administration as acombination therapy in conjunction with the antibodies described hereinare modulators (e.g., agonists and antagonists) of certain members ofthe TGFβ super family of growth factors, such as BMP6, BMP7, GDF11,TGFβ2, TGFβ3, RGMc, etc.

Any of the above-mentioned agents can be administered in combinationwith the myostatin antibody of the invention to treat a metabolicdisease, or a disease associated with an impaired neurological signalingbetween a neuron and a target tissue, e.g., spinal cord injury, muscularatrophy, and muscular dystrophy.

Use of Anti-Pro/Latent-Myostatin Antibodies or Antigen Binding FragmentsThereof for Treating Diseases/Disorders

Pharmaceutical compositions described herein are suitable foradministration to human patients for the treatment or prevention ofdiseases and conditions where reduced myostatin signaling is desirable.Such diseases and conditions include, but are not limited to: metabolicdisorders, and diseases associated with impaired neurological signaling,e.g., spinal cord injury. Exemplary conditions for which thecompositions and methods of the present invention may be useful arefurther described below.

A. Metabolic Disorders and Diseases

The invention provides methods for treating or preventing a metabolicdisease in a subject. As used herein, the term “metabolic disease”refers to any undesirable condition involving perturbation of the normalphysiological state of homeostasis due to an alteration in metabolism(anabolism and/or catabolism). Metabolic disorders affect how the bodyprocesses substances needed to carry out physiological functions and aregenerally associated with aberrant glucose, lipid/fat and/orprotein/nitrogen metabolism, or osmotic dysregulation, and pathologicalconsequences arising from such condition. A number of metabolicdisorders of the invention share certain characteristics, e.g., they areassociated with a loss of fat-free or lean muscle mass, an excess of fatmass, a lower metabolic rate, insulin resistance, lack of ability toregulate blood sugar, weight gain, and/or increase in body mass index.In some cases, such metabolic conditions may be triggered or exacerbatedby medication that the patients receive. As discussed in more detailherein, metabolic disorders can occur secondarily to, or occur as aresult of, a muscle condition or disorder.

The present invention is based, at least in part, on the discovery thatadministration of a myostatin inhibitor, e.g., an antibody, or antigenbinding fragment thereof, that specifically binds to pro/latentmyostatin, to subjects having a metabolic disease significantly improvesboth the physiological and the functional characteristics of the injuredsubjects. In particular, the present inventors have surprisinglydiscovered that administration of a myostatin inhibitor, e.g., ananti-myostatin antibody or antigen-binding portion thereof,significantly increases the metabolic rate or energy expenditure insubjects having metabolic disease. Administration of a myostatininhibitor, e.g., an anti-myostatin antibody or antigen-binding portionthereof, also significantly attenuated SCI-induced reduction insub-lesional muscle mass and overall body mass and, while at the sametime reducing the mass of undesirable adipose tissue such as white andvisceral adipose tissue. In addition, subjects who received a myostatininhibitor, e.g., an anti-myostatin antibody or antigen-binding portionthereof, treatment exhibited a significant improvement in theirlocomotor function, muscle strength, as well as motor coordination andbalance skills.

Accordingly, the present invention provides methods for treating orpreventing metabolic diseases in a human subject. The methods includeselecting a human subject suffering from a metabolic disease, andadministering to the human subject an effective amount of a myostatininhibitor, e.g., an antibody, or antigen binding fragment thereof, thatspecifically binds myostatin, thereby treating or preventing themetabolic disease in the human subject. Preferably, the antibody, orantigen binding fragment thereof, specifically binds to pro/latentmyostatin, but docs not bind to GDF11. Antibodies that specificallyrecognize pro/latent myostatin, but not GDF11, are beneficial and avoidundesirable toxicity caused by off-target binding of antibodies to GDF11in the subject. In one embodiment, the subject is a pediatric subject.

Examples of metabolic diseases that may be treated or prevented by themethods of the present invention include but are not limited to, type 1diabctcs, type 2 diabctcs, metabolic syndrome, pre-diabetes, obesity,cardiovascular diseases (such as congestive heart failure),non-alcoholic stetohepatitis (NASH), spinal cord injury (SCI) (e.g.,complete or incomplete/partial SCI), hypo-metabolic states, doublediabetes, Cushings disease (also referred to as Cushing's syndrome),obesity syndrome (e.g., diet-associated or diet-induced obesity),insulin resistance, insulin insufficiency, hyperinsulinemia, impairedglucose tolerance (IGT), abnormal glycogen metabolism, hyperlipidemia,hypoalbuminemia, hypertriglyceridemia, syndrome X, fatty liver diseaseand metabolic bone diseases. In some embodiments, metabolic diseasesinclude diseases associated with impaired neurological signaling orpartial denervation. In some embodiments, metabolic diseases includeconditions triggered by or associated with certain medication (e.g.,side effects).

Additional diseases or conditions related to metabolic disorders and/orbody composition that would be apparent to the skilled artisan and arewithin the scope of this disclosure.

Diabetes refers to a group of metabolic diseases characterized by highblood sugar (glucose) levels which result from defects in insulinsecretion or action, or both. There are two most common types ofdiabetes, namely type 1 diabetes and type 2 diabetes, which both resultfrom the body's inability to regulate insulin. Insulin is a hormonereleased by the pancreas in response to increased levels of blood sugar(glucose) in the blood.

The term “type 1 diabetes,” as used herein, refers to a chronic diseasethat occurs when the pancreas produces too little insulin to regulateblood sugar levels appropriately. Type 1 diabetes is also referred to asinsulin-dependent diabetes mellitus, IDDM, and juvenile onset diabetes.People with type I diabetes (insulin-dependent diabetes) produce littleor no insulin at all. Although about 6 percent of the United Statespopulation has some form of diabetes, only about 10 percent of alldiabetics have type I disorder. Most people who have type I diabetesdeveloped the disorder before age 30. Type 1 diabetes represents is theresult of a progressive autoimmune destruction of the pancreatic β-cellswith subsequent insulin deficiency. More than 90 percent of theinsulin-producing cells (beta cells) of the pancreas are permanentlydestroyed. The resulting insulin deficiency is severe, and to survive, aperson with type I diabetes must regularly inject insulin.

In type II diabetes (also referred to as noninsulin-dependent diabetesmellitus, NDDM), the pancreas continues to manufacture insulin,sometimes even at higher than normal levels. However, the body developsresistance to its effects, resulting in a relative insulin deficiency.Type 11 diabetes may occur in children and adolescents but usuallybegins after age 30 and becomes progressively more common with age:about 15 percent of people over age 70 have type II diabetes. Obesity isa risk factor for type II diabetes, and 80 to 90 percent of the peoplewith this disorder are obese.

In some embodiments, diabetes includes pre-diabetes. “Pre-diabetes”refers to one or more early diabetic conditions including impairedglucose utilization, abnormal or impaired fasting glucose levels,impaired glucose tolerance, impaired insulin sensitivity and insulinresistance. Prediabetes is a major risk factor for the development oftype 2 diabetes mellitus, cardiovascular disease and mortality. Muchfocus has been given to developing therapeutic interventions thatprevent the development of type 2 diabetes by effectively treatingprediabetes.

In some embodiments, diabetes includes double diabetes, which is acombination of type 1 diabetes with features of insulin resistance andtype 2 diabetes.

Diabetes can be diagnosed by the administration of a glucose tolerancetest. Clinically, diabetes is often divided into several basiccategories. Primary examples of these categories include, autoimmunediabetes mellitus, non-insulin-dependent diabetes mellitus (type 1NDDM), insulin-dependent diabetes mellitus (type 2 IDDM), non-autoimmunediabetes mellitus, non-insulin-dependent diabetes mellitus (type 2NIDDM), and maturity-onset diabetes of the young (MODY). A furthercategory, often referred to as secondary, refers to diabetes broughtabout by some identifiable condition which causes or allows a diabeticsyndrome to develop. Examples of secondary categories include, diabetescaused by pancreatic disease, hormonal abnormalities, drug- orchemical-induced diabetes, diabetes caused by insulin receptorabnormalities, diabetes associated with genetic syndromes, and diabetesof other causes. (see e.g., Harrison's (1996) 14th ed., New York,McGraw-Hill).

Obesity is another prevalent metabolic disease that can be treated orprevented by the methods of the present invention. “Obesity” refers to achronic condition defined by an excess amount body fat. The normalamount of body fat (expressed as percentage of body weight) is between25-30% in women and 18-23% in men. Women with over 30% body fat and menwith over 25% body fat are considered obese. Obesity can be definedusing any clinically relevant definitions. For example, in adults, bodymass index (BMI, kg/m²) is frequently used as a measure of overweightand obesity, with overweight being defined as a BMI 25-29.9 kg/m²,obesity as a BMI equal to or greater than 30 kg/m², and morbid obesitybeing defined as BMIs over 40 kg/m². Obesity can also be defined inadults by central adiposity as measured by waist circumference, withraised waist circumference defined as equal to or greater than 102 cm inmen and equal to or greater than 88 cm in women. Subject with obesitymay exhibit other symptoms such as increased fasting plasma glucose,increased fasting plasma triglycerides, decreased fasting high densitylipoprotein (HDL) level, and increased blood pressure. Obesity may alsocause various orthopedic problems, skin disorders and swelling of thefeet and ankles Severe complications of obesity include a much higherrisk of coronary artery disorder and of its major risk factors type IIdiabetes, hyperlipidemia and hypertension. Much of the morbidityassociated with obesity is associated with type II diabetes, as poorlycontrolled diabetes and obesity lead to a constellation of symptoms thatare together known as syndrome X, or metabolic syndrome. In someembodiments, the obesity is sarcopenic obesity. In some embodiments, thesubject having obesity is on a caloric restriction regimen.

The methods of the present invention are also suitable for treating orpreventing metabolic disease such as metabolic syndromes. As usedherein, “metabolic syndrome” refers to the concept of a clustering ofmetabolic risk factors that come together in a single individual andlead to a high risk of developing diabetes and/or cardiovasculardiseases. The main features of metabolic syndrome include insulinresistance, hypertension (high blood pressure), cholesterolabnormalities, dyslipidemia, triglyceride abnormalities, an increasedrisk for clotting and excess body weight, especially in the abdomen, orobesity. The American Heart Association suggests that metabolic syndromebe diagnosed by the presence of three or more of the followingcomponents: (1) an elevated waist circumference (men, equal to orgreater than 40 inches (102 cm); women, equal to or greater than 35inches (88 cm)); (2) elevated triglycerides (equal to or greater than150 mg/dL); (3) reduced High Density Lipoprotein cholesterol or HDL(men, less than 40 mg/dL; women, less than 50 mg/dL); (4) elevated bloodpressure (equal to or greater than 130/85 mm Hg); and (5) elevatedfasting glucose (equal to or greater than 100 mg/dL).

In another aspect, the methods of the present invention are suitable fortreating or preventing metabolic disease such as obesity syndromes. Theterm “obesity syndrome” refers any disorder or conditions causing asubject to be grossly fat or overweight. Like other metabolic diseases,people with obesity syndrome are usually associated a loss of fat-freeor lean muscle mass, an excess of fat mass, a lower metabolic rate,insulin resistance, lack of ability to regulate blood sugar, weightgain, and increase in body mass index. In some embodiments, the obesitysyndrome is selected from the group consisting of Prader Willi, anobesity syndrome associated with a genetic disorder, and an obesitysyndrome associated with a hypothalamic disorder.

The methods of the present invention are also suitable for treating orpreventing metabolic diseases associated with a hypo-metabolic state.The term “a hypo-metabolic state” refers to a state of reducedmetabolism or metabolic activity, where the body is not producing enoughenergy. Patients with a hypo-metabolic state generally have a lowermetabolic rate, a loss of fat-free or lean muscle mass, an excessivegain of fat mass, insulin resistance, lack of ability to regulate bloodsugar, weight gain, and an increase in body mass index. In someembodiments, the hypo-metabolic state is selected from the groupconsisting of a state associated with prolonged immobilization, a stateassociated with bed-rest, a state associated with casting, a stateassociated with a stroke, a state associated with amputation, and apost-surgery state. In some embodiments, the hypo-metabolic state is apost-surgery state, e.g., paraspinal muscle atrophy after lumbar spinesurgery. In one embodiment, the paraspinal muscle atrophy is a nerveinjury-dependent muscle atrophy. In one embodiment, the surgery is aspinal surgery. In one embodiment, the spinal surgery is a lumbar spinesurgery or a lumbar spine procedure, e.g., a lumbar fusion procedure, alumbar nonfusion procedure, a posterior lumbar fusion procedure, ananterior lumbar fusion procedure, a minimally invasive (MIS) posteriorlumbar decompression procedure, a minimally invasive (MIS) posteriorlumbar fusion procedure, a non-MIS equivalent procedure, etc.

In another aspect, the methods of the present invention are suitable fortreating or preventing metabolic diseases such as Cushings disease,which is also referred to as Cushing's syndrome or Cushing syndrome. Theterm “Cushings disease” refers to a collection of signs and symptoms dueto prolonged exposure to cortisol. This may stem from endogenous causes,such as a condition in which the pituitary gland releases too muchadrenocorticotropic hormone (ACTH), or exogenous causes, such as the useof oral corticosteroid medication. Some of the hallmark signs andsymptoms of Cushing disease may include: progressive obesity, such asweight gain and fatty tissue deposits, particularly around themidsection and upper back and between the shoulders (buffalo hump)(upper body obesity above the waist); thin arms and legs, round, red,full face (moon face); changes in the skin, such as pink or purplestretch marks (striae) on the skin of the abdomen, thighs, breasts andarms, thinning, fragile skin that bruises easily, low healing of cuts,insect bites and infections, and acne. Patients with Cushing disease mayalso experience severe fatigue, muscle weakness, depression, anxiety andirritability, loss of emotional control, cognitive difficulties, new orworsened high blood pressure, headache, type 2 diabetes, and/or honeloss which may lead to fractures over time. In children, Cushing diseasemay cause impaired growth (slow growth rate). In some embodiments, theCushings disease is selected from the group consisting ofcorticosteroid-induced Cushings disease and tumor-induced Cushingsdisease.

To date, standard treatments for Cushing disease are designed to lowerthe high level of cortisol in the body, whether the source is endogenousoverproduction of the hormones or due to medication. The best treatmentfor a particular patient depends on the cause of the syndrome. Treatmentoptions that are currently available include, for example, reducingcorticosteroid use, surgery, radiation therapy, and medications.

Thus, the use of inhibitors of myostatin activation described hereinpresents an alternative or additive treatment option for patientssuffering from Cushing's disease.

Where the cause of Cushing disease is long-term use of corticosteroidmedications, controlled reduction of the dosage of the drug over aperiod of time, while still adequately managing the underlining diseaseor condition for which the drug is being administered, may beconsidered. Thus, in some embodiments, the patient who is on acorticosteroid therapy has one or more autoimmune or inflammatorydiseases, such as rheumatoid arthritis, lupus and asthma. Corticosteroidmay also be prescribed to patients to suppress the body's immunity inorder to prevent the body from rejecting an allograft transplant, suchas a transplanted organ or tissue.

In some embodiments, patients receiving a corticosteroid therapy includea sub-population of individuals who do not tolerate well, who are poorlyresponsive or not responsive, to other treatment options, such asnon-corticosteroid medications. In such situations, the physician maycontinue to prescribe corticosteroid medication. In some embodiments,surgery may be considered as an alternative option.

If the cause of Cushing syndrome is a tumor, complete surgical removaland/or radiation therapy can be considered. In some embodiments,patients have a tumor in the pituitary, adrenal glands, lungs orpancreas. After the operation, cortisol replacement therapy is typicallyadministered to provide the body with the correct amount of adrenalhormone production.

In some embodiments, patients with Cushing syndrome never experience aresumption of normal adrenal function and therefore may require lifelongreplacement therapy. The inhibitors of myostatin activation describedherein may be suitable to treat such patients.

In some embodiments, medication can be used to control cortisolproduction when surgery and/or radiation don't work. Medications mayalso be used before surgery in patients who have become very sick withCushing syndrome. The inhibitors of myostatin activation encompassed bythe present disclosure may be used to treat such patients prior tosurgery to improve signs and symptoms and minimize surgical risk.

Medications currently used to control excessive production of cortisolat the adrenal gland include ketoconazole (Nizoral), mitotane (Lysodren)and metyrapone (Metopirone). Mifepristone (Korlym) is approved forpatients with Cushing syndrome who have type 2 diabetes or glucoseintolerance. Mifepristone does not decrease cortisol production, but itblocks the effect of cortisol on the tissues. Side effects from thesemedications may include fatigue, nausea, vomiting, headaches, muscleaches, high blood pressure, low potassium and swelling. Some have moreserious side effects, such as neurological side effects and livertoxicity. The inhibitors of myostatin activation encompassed by thepresent disclosure may be used alone (in lieu of) or in combination withany of these therapeutics.

More recently, pasireotide (Signifor) has become available for thetreatment of Cushings, which works by decreasing ACTH production from apituitary tumor. This medication is given as an injection twice daily.It is typically recommended when pituitary surgery is unsuccessful orcannot be done. Side effects associated with this medication are fairlycommon, and may include diarrhea, nausea, high blood sugar, headache,abdominal pain and fatigue. The inhibitors of myostatin activationencompassed by the present disclosure may be used alone (in lieu of) orin combination with any of such therapeutics.

In some embodiments, the tumor or its treatment will cause otherhormones produced by the pituitary or adrenal gland to become deficient,which may require hormone replacement therapy. In some embodiments, noneof these currently available treatment options are appropriate oreffective, surgical removal of the adrenal glands (bilateraladrenalectomy) may be considered, which will require lifelongreplacement medications. Patients who are candidates for such option maybenefit from a myostatin inhibition therapy described herein, beforeand/or after adrenalectomy.

In yet another aspect, the methods of the present invention are suitablefor treating or preventing metabolic diseases such as cardiovasculardisease. The term “cardiovascular disease” refers to any disease of theheart or blood vessels. Cardiovascular or heart disease includes but isnot limited to, for example, angina, arrhythmia, coronary artery disease(CAD), coronary heart disease, cardiomyopathy (including dilatedcardiomyopathy, restrictive cardiomyopathy, arrhythmogenic rightventricular cardiomyopathy, and diabetic cardiomyopathy), heart attack(myocardial infarction), heart failure (e.g., CHF), hypertrophiccardiomyopathy, mitral regurgitation, mitral valve prolapse, pulmonarystenosis, etc. Blood vessel disease includes but is not limited to, forexample, peripheral vascular disease, artery disease, carotid arterydisease, deep vein thrombosis, venous diseases, and atherosclerosis. Insome embodiments, a subject having heart failure is resistant todiuretic therapy. In another embodiment, a subject having heart failureresponds poorly to diuretic therapy.

Another aspect of the disclosure includes a method of treating a subjecthaving a metabolic disease or condition related to aging. Exemplarydiseases and conditions related to ageing include, without limitation,sarcopenia (age-related muscle loss), frailty, and androgen deficiency.

Another aspect of the disclosure includes a method of treating a subjecthaving a metabolic disease or condition related to disuse or geneticatrophy/trauma, e.g., atrophy caused by disuse, atrophy caused bygenetic mutation(s), atrophy resulting from an injury. Exemplary suchdiseases and conditions include, without limitation, muscle weaknessrelated to time spent in an intensive care unit (ICU), hip/jointreplacement, hip fracture, stroke, bed rest, SCI, rotator cuff injury,knee replacement, bone fracture, and burns.

The present disclosure includes beneficial effects of myostatininhibition on bone homeostasis. In the musculoskeletal system (definedas the bones of the skeleton, muscles, cartilage, tendons, ligaments,joints, and other connective tissue that supports and binds tissues andorgans together), the homeostasis of muscle and bone is intimatelyconnected. Bone grows in response to muscle growth is a mechanosensitiveprocess that is regulated by endocrine signaling.

Like muscle, bone homeostasis involves a dynamic processes of balancingbone growth (bone formation) and bone loss (bone resorption). Parametersthat may be used to assess bone homeostasis include but are not limitedto: bone mass, volume, density, cross section area, strength, frequencyof fractures, rate of bone repair, etc. Factors (e.g., cytokines,hormones) that are known to play a role in this process include, but arenot limited to: parathyroid hormone, 1,25-dihydroxyvitamin D3, T4,corticosteroids, prostaglandins such as prostaglandin E2, Interleukin-4,Interleukin-18, Interferon-γ, Interleukin-17, Interleukin-6,Interleukin-1, RANKL, CSFs, TGFβ, osteoprotegerin, BMPs, TGFs and FGFs.Osteoclasts and osteoblasts contribute to bone resorption and growth,respectively.

From a structural point of view, bone strength is determined bycombination of trabecular and cortical bone architecture andcomposition. Generally, decreased bone strength is observed with age,e.g., osteopenia and osteoporosis. In some circumstances, bone loss isdue to other factors, such as medication. For example, glucocorticoidtreatment may cause bone loss. Medical conditions that may be associatedwith bone loss include, without limitation, cancer and muscle/metabolicdisorders with comorbid bone loss. In some embodiments, such conditionsare associated with spinal cord injury (SCI), muscular dystrophy such asDMD, obesity, and/or Cushing disease.

Bone loss can be measured using assays well known to one of ordinaryskill in the art (sec Shanmugarajan et al., J. Pathol., 2009:219(1):52-60 and Wasserman et al., Neuromuscular Disorders, 2017,27(4):331-337). For example, bone mineral density, e.g., areal bonemineral density, can be measured using a DXA scan of the lumbar spine,whole body, and lateral distal femur in accordance with ISCDrecommendations (Wasserman et al., Neuromuscular Disorders, 2017,27(4):331-337). After scanning, bone mineral density can be calculatedusing reference data from Henderson et al., Am. J. Roentgenol, 2002,178:439-443; Kalkwarf et al., J. Bone Miner. Res., 2013, 28:206-212;Kelly et al., J. Pediatr. Hematol. Oncol., 2005, 27:248-253; and Zemelet al., J. Clin. Endocrinol. Metab., 2011, 96:3160-3169. Fracturehistory of patients can also be collected, including age at fracture,number of fractures, and location of fractures. Osteoporosis istypically measured using the 2013 ISCD criteria, which includesvertebral compression fractures in the absence of high energy trauma orinfiltrative disease; or a BMD Z-score of ≤2.0 SD, and two or more longbone fractures by 10 years of age, or three or more long hone fracturesby 19 years of age (see, e.g., Bishop et al., J. Clin. Densitom, 2014,17:275-280).

Several therapeutics are currently available for the treatment of boneloss. Agents used to slow the rate of bone loss include Bisphosphenatesand Denosumab. Bisphosphenates block osteoclast recruitment and induceosteoclast apoptosis. These are often used in treatment ofpost-menopausal and glucocorticoid induced osteoporosis, Paget'sdisease, and malignant hypercalcemia. Denosumab, developed by Amgen, isa monoclonal antibody that blocks RANKL (osteoclast development). It isused to treat osteoporosis, metastases to bone, other bone tumors.

Agents used to grow new bone include Teriparatide and Romosozumab.Forteo is a Teriparatide marketed by Eli Lilly, which is a recombinantprotein fragment comprising the first 34 amino acid residues ofparathyroid hormone. It is typically used to treat osteoporosis, andpatients who are at high risk for bone fracture, as well as patients whoare intolerant to other therapies. Romosozumab, available from Amgen, isa monoclonal antibody that blocks sclerostin (a Wnt pathway antagonist).To date, there is no drug that can increase both bone and muscle.

Several groups are carrying out preclinical and clinical studies withagents that at least in part affect the myostatin pathway. For example,Acceleron has developed ActRIIA and ActRIIB ligand trap agents (e.g.,ACE-011, ACE-536, ACE-031 and ACE-2494), at least some of which are saidto increase bone mass when administered in vivo. None of these agentsappears to be specific to myostatin/GDF8 but also affects one or more ofthe other pathways. Eli Lilly's monoclonal antibody LY2495655(Landogrozumab) did not increase bone mass in humans undergoing electivehip replacement, as measured by dexa. This antibody binds both GDF8 andGDF11.

Unlike these agents that affect myostatin as well as additionalpathway(s), monoclonal antibodies encompassed by the present disclosurespecifically bind and inhibit the activation step of myostatin/GDF8. Insome embodiments, such antibodies bind proMyostatin and/or latentmyostatin, thereby inhibiting activation and subsequent release ofmature myostatin, but do not bind mature myostatin that is notassociated with a latent (inactive) complex. In some embodiments, theantibodies or fragments thereof bind tethered forms (e.g.,intramuscular) of inactive myostatin (e.g., pro-myostatin), which havethe ability to locally act upon tissue-associated myostatin within adisease niche. In some embodiments, the antibodies or fragments thereofbind soluble forms (e.g., in circulation) of inactive myostatin (e.g.,latent-myostatin), which have the ability to act upon circulating latentmyostatin that may have endocrine or systemic effects. In any of suchembodiments, preferred inhibitors of myostatin for carrying out themethods of the present invention are those that are selective formyostatin that do not antagonize other members of the TGFβ superfamilyof growth factors/cytokins, such as GDF11. Such selectivity isadvantageous particularly in pediatric patient populations and/orpatient populations requiring a long-term care (e.g., chronic therapy),where inhibiting other pathways, such as GDF11, may produce harmful orunwanted side effects or adverse events. The inventors of the presentdisclosure have shown that such antibodies can effectively inhibitmyostatin activation and cause beneficial muscle effects and metaboliceffects. Furthermore, evidence provided herein shows that suchantibodies can also cause beneficial biological effects on bonehomeostasis in viva (see FIGS. 28-31).

Accordingly, the present invention includes the use of an inhibitor ofmyostatin activation for enhancing one or more parameters of bonehomeostasis, including: relative bone volume (e.g., as measured by bonevolume over total volume of the corresponding tissue or sample);trabecular bone volume, trabecular number; trabecular thickness;trabecular spacing/separation; bone cross section (e.g., as measured bycortical cross sectional area); cortical bone area; cortical endostealperimeter; cortical periosteal perimeter; cortical porosity; andcortical cross section thickness. In some embodiments, the inhibitor ofmyostatin activation described herein can produce a clinicallymeaningful bone effects. In some embodiments, administration of theinhibitor causes at least a 10% increase in one or more of theparameters listed above, e.g., at least 10%, at least 11%, at least 12%,at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, atleast 18%, at least 19%, at least 20%, at least 21%, at least 22%, atleast 23%, at least 24%, and at least 25%.

The present invention also includes the use of an inhibitor of myostatinactivation for increasing one or more of these parameters inweight-hearing hone or in non-weight hearing hone. As discussed in moredetail in the Examples, weight-bearing activity is an important stimulusfor bone mass accrual. Surprisingly, a myostatin inhibitor may be usedto not only enhance bone parameters in weight-bearing bone, but may alsobe used to enhance these parameters in non-weight bearing bone. Clearly,weight-bearing and non-weight-bearing bones differ among species. Inrodents, for example, non-weight-bearing bone includes the vertebrae. Anincrease in non-weight bearing bone parameters further demonstrates thatmyostatin inhibitors disclosed herein not only act to increase bonethrough, for example, increased muscle stimulation, but also act as akey regulator to increase the general metabolism and bone health of thetreated animals.

Thus, preferred inhibitors of myostatin activation described herein arecharacterized by two or more of the following attributes: a) the abilityto enhance muscle mass, b) the ability to prevent muscle loss, c) theability to enhance motor function, d) the ability to prevent orameliorate metabolic dysregulation, e) the ability to enhance bone mass,f) the ability to reduce bone loss, g) the ability to increase bonemineral density; without directly inhibiting any other members of theTGFβ super family of growth factors, such as GDF11 and Activin.

Accordingly, such an inhibitor can be used in human patients for: i) theprevention of bone fracture (e.g., reducing the frequency of suchincidents and/or severity or degree of fracture); ii) the treatment ofbone fractures (e.g., to facilitate bone healing, growth orregeneration); and enhancement of bone strength (to strengthen weakenedbones, such as age-related, injury-associated or disease-associated). Insome embodiments, such use can be combined with additional agent(s)intended to enhance bone (bone-enhancing or bone-protective agents),such TGFβ antagonists (preferably TGFβ1 inhibitors), bisphosphonates,calcium, vitamin D, RANKL inhibitors, etc.

In some embodiments, suitable patient populations include those withCushing's syndrome.

B. Diseases Associated with Impaired Neurological Signaling

The present disclosure is based, at least in part, on the surprisingdiscovery that inhibition of myostatin signaling may be particularlyuseful for the intervention of conditions involving defects incommunication between muscle and its innervating neurons. The findingspoint to a close coordination/relationship between the musculoskeletalsystem and the nervous system. Needless to say, the spinal cord housesmajor nerves that control motor function. Thus, the disclosure providesmethods for treating or preventing diseases associated with impairedneurological signaling between a neuron and a target tissue thatexpresses myostatin in subjects, e.g., human subjects. A disorder maybe, for example, injury-based (e.g., a spinal cord injury) or genetic(e.g., resulting from a genetic mutation, e.g., SMA).

In some embodiments, the methods include administering to a subjectsuffering from a disease associated with an impaired neurologicalsignaling between a neuron and a target tissue an effective amount of amyostatin inhibitor, e.g., an antibody, or antigen binding fragmentthereof, that specifically hinds myostatin and inhibits myostatinsignaling, thereby treating or preventing the disease associated withthe impaired neurological signaling in the subject. Preferably, theantibody, or antigen binding fragment thereof, specifically binds topro/latent myostatin, but does not bind to mature GDF11. In someembodiments, such antibody or fragment does not bind maturemyostatin/GDF8.

As used herein, term “disease with an impaired neurological signaling”refers to any disease or disorder that is caused by, or associated with,a disrupted signal transduction or a breakdown in communication betweena neuron and its target tissue(s), e.g., a muscle tissue, a braintissue, a liver tissue, a blood vessel tissue, or an adipose tissue. Insome embodiments, the impaired neurological signaling occurs due to adamage in the neuron structure, where neurons are incapable oftransmitting signals towards their targets. In other embodiments, thestructures of neurons remain intact, but there are functional disruptionor defects, for example, a blockage at the neuromuscular junction, suchthat the ability of neurons to transmit signals is affected.

In some embodiments, “disease with an impaired neurological signaling”refers to disease or condition associated with denervation, e.g., apartial loss or perturbation of nerve supply or neuronal input to itstarget, such as muscle. In some embodiments, denervation is induced byinjury. In some embodiments, denervation is associated with a disease,such as a genetic disease. In cases of genetic diseases, in someembodiments, the patient may be diagnosed with the genetic disease bygenetic screening. In some embodiments, such genetic screening may beperformed in a fetal, neonatal or pediatric subject. Non-limitingexamples of diseases with an impaired neurological signaling include,for example, vocal cord paresis/paralysis, spinal cord injury (SCI),myasthelia gravis, amyotrophic lateral sclerosis (ALS), and spinalmuscular atrophy (SMA).

Spinal Cord Injury

The methods of the present invention are also suitable for treating orpreventing conditions with an impaircd neurological signaling due tonerve injury. In some embodiments, such condition is spinal cord injury(SCI). As used herein, the term “spinal cord injury” refers to damagesto any part of the spinal cord or nerves at the end of the spinal canal.Spinal cord injury often causes permanent changes in strength, sensationand other body functions below the site of the injury. Each year, it isestimated that there are 12,500 new cases of spinal cord injury (US).Prevalence is 275,000 cases in the US and roughly 60% have paraplegia.There are no therapies in development directed at reversing or reducingmuscle atrophy in SCI and this represents a large unmet need. Whilethere is significant patient heterogeneity based on time since injury,level and completeness of injury, and extent of disability, physicalrehabilitation to improve muscle function and metabolic outcomes isstandard of care.

SCI patients are stratified based on the level (paraplegia vs.tetraplegia) and the completeness of the lesion (complete vsincomplete). This stratification has been developed into the ASIA scale,with two broad groups based on level of paralysis: complete (ATS gradesA/B) and incomplete (ATS grades C/D/E), defined below:

A: Complete motor and sensory loss

B: Motor loss with retained sensory perception (still can feel touch,pressure)

C and D: Incomplete motor loss

E: Most function is regained: this represents a low proportion of thepopulation.

There are 7 cervical (neck), 12 thoracic (chest), 5 lumbar (back), and 5sacral (tail) vertebrae. A lesion in SCI may occur at any location alongthe vertebrae. The key muscles that need to be tested to establishneurologic level are as follows:

-   -   C5: Elbow flexors (biceps, brachialis)    -   C6: Wrist extensors (extensor carpi radialis longus and brevis)    -   C7: Elbow extensors (triceps)    -   C8: Long finger flexors (flexor digitorum profundus)    -   T1: Small finger abductors (abductor digiti minimi)    -   L2: Hip flexors (iliopsoas)    -   L3: Knee extensors (quadriceps)    -   L4: Ankle dorsiflexors (tibialis anterior)    -   L5: Long toe extensors (extensor hallucis longus)    -   S1: Ankle plantar flexors (gastrocnemius, soleus)

With a complete spinal cord injury, the cord can't send signals belowthe level of the injury. As a result, patients are paralyzed below theinjury. With an incomplete injury, patients will have some movement andsensation below the injury.

There are multiple phases associated with spinal cord injury. Subjectsmay be in an acute spinal cord injury phase immediately after injury,where diagnosis between complete and incomplete injury is generallydifficult, due in part to the trauma and associated inflammation.Typically, the acute phase is defined as the initial in-hospital periodfollowing the event/injury in acutc medical/surgical care, which isgenerally around ˜2 weeks. A subject may be in a sub-acute spinal cordinjury phase, where there is a distinction between complete andincomplete spinal cord injury, and recovery is possible through ongoingrehab. Typically the sub-acute phase constitutes ˜2 weeks up to ˜18months post injury (e.g., 3-6 months post-injury). Yet further, asubject may be in a chronic spinal cord injury phase which generallystarts around 6-12 months after the time of injury, where patients havedemonstrated substantial decrease in rate of recovery or when rehabefforts have reached a stable phase (e.g., plateau) despite the ongoingstandard of care efforts.

Muscle strength always should be graded according to the maximumstrength attained, no matter how briefly that strength is maintainedduring the examination. The muscles are tested with the patient supine.Motor level is determined by the most caudal key muscles that havemuscle strength of 3 or above while the segment above is normal (=5).

Motor index scoring uses the 0-5 scoring of each key muscle, with totalpoints being 25 per extremity and with the total possible score being100.

Lower extremities motor score (LEMS) uses the ASIA key muscles in bothlower extremities, with a total possible score of 50 (i.e., maximumscore of 5 for each key muscle [L2, L3, L4, L5, and S1] per extremity).A LEMS of 20 or less indicates that the patient is likely to be alimited ambulator. A LEMS of 30 or more suggests that the individual islikely to be a community ambulator.

ASIA recommends use of the following scale of findings for theassessment of motor strength in spinal cord injury:

-   -   0: No contraction or movement    -   1: Minimal movement    -   2: Active movement, but not against gravity    -   3: Active movement against gravity    -   4: Active movement against resistance    -   5: Active movement against full resistance

Monitoring functional outcomes and quality of life in SCI patients is acomplex task as selection of the appropriate functional measure dependsupon the completeness and level of injury. One common measure which isapplicable to all patients is the functional independence measure (FIM)which is a 7-point scale designed to quantify the dependence of apatient on a caregiver. An additional metric for measuring quality oflife which has had recent attention is the SCI-QOL, which integratesboth functional skills and emotional health of the patient (Tulsky 2015,J Spinal Cord Med. 38(3): 257-69). Many other functional outcomemeasures have been outlined by the SCIRE project.

In some embodiments, meaningful clinical effects achieved byadministration of an effective amount of the myostatin inhibitordescribed herein to SCI patients may correspond to at least a 6 point(≥6) increase from baseline in total motor score of ASIA at, e.g., week24. In some embodiments, meaningful clinical effects achieved byadministration of an effective amount of the myostatin inhibitordescribed herein to SCI patients may correspond to statisticallysignificant difference in the meantotal SCIM III score between treatedand untreated/control groups at Day 112 (+/−7 days). In someembodiments, meaningful clinical effects achieved by administration ofan effective amount of the myostatin inhibitor described herein to SCIpatients may correspond to greater than a 4 point (>4) increase inFunctional Independence Measure for Locomotion (FIM-L) score.

Individuals with spinal cord injury have an increased prevalence ofabnormalities in carbohydrate and lipid metabolism associated withimmobilization, muscle atrophy, and increased adiposity. The bodycomposition is substantially altered and typified by rapid and long-termdecline in metabolically active muscle mass and bone with starkincreases in central adiposity. The latter contributes to a maladaptivemetabolic profile favoring substantial gain in body mass occurring 2-7months following injury. Occurring together these co-morbid risk factorsincite all-cause cardiovascular disease, diabetes, and risk clusteringas cardiometabolic disease, the latter including component hazards fordyslipidemia, glucose intolerance and insulin resistance.

Rapid and profound muscle wasting affects those with a spinal cordinjury and impacts the entire body, not just the denervated limbs.Muscle loss is believed to be due to a combination of factors includingdenervation (of the paretic limbs), immobilization, inflammation,factors released by the paralyzed muscle, steroid use, infections, andlack of nutrition. A large percentage (˜30%) of lean muscle mass is lostin the first six weeks following injury (the acute phase). Thisaccelerated rate of lean mass loss continues on into chronic conditionswith a decrease in lean mass (per decade) of 3% for tetraplegia and 2.4%for paraplegia (as compared to a decline of 1% seen in healthy controls)(Spungen 2003). This accelerated muscle atrophy contributes to prematuresarcopenia.

An SCI patient experiences profound changes in total body composition.In particular, lean muscle mass is replaced with fat mass, on average anSCI patient has 13% more fat tissue per unit SMI than a healthy control,with a significant increase in intramuscular fat (Spungen 2003, Gorgey2007). This whole-body change in composition (˜60-70% are obese) hasprofound impacts on metabolism which is evidenced by increasedprevalence of cardiovascular disease, type II diabetes, and thyroiddisorders.

Mechanical unloading following spinal cord injury also translates intodisruptions in bone homeostasis. SCI patients have reduced bone mineralcontent, develop osteoporosis, and suffer from increased rates offractures (as many as 50% of SCI patients will experience a fracturepost injury) (Battaglino 2013). A fracture leads to hospitalization andcan have profound consequences by increasing the risk for developingpressure ulcers, contractures of the knee and hip, and for experiencinga hypertensive crisis.

Overall increases in lean mass and decrease in fat mass in SC1 patientscan be monitored by several well-validated methods, such as thigh orupper arm muscle volume by magnetic resonance imaging, or total bodycomposition by dual-energy x-ray absorptiometry or DEXA. Suchmeasurements are routinely performed in the field.

Outcome or progress of therapy (e.g., overall clinical effects) may bemeasured by using any of well-characterized tests commonly employed forevaluating SCI clinical practice. These tests are useful for i)providing information on each measure's clinical utility andpsychometric properties; ii) assisting clinicians to select appropriatemeasures tailored to particular patient(s); iii) identifying individualswho may benefit from a certain therapy; iv) monitoring progress; v)evaluating whether treatments are effective; and/or, vi) help programsimprove services to patients and medical professionals. Suitableclinical evaluation tools/tests available for patients include, but arenot limited to the following:

For evaluating Assistive Technology, useful tests include: AssistiveTechnology Device Predisposition Assessment (ATD-PA); Quebec UserEvaluation of Satisfaction with Assistive Technology (QUEST 2.0); andWingate Anaerobic Testing (WAnT).

For evaluating Community Reintegration, useful tests include: Assessmentof Life Habits Scale (LIFE-H); Community Integration Questionnaire(CIQ); Craig Handicap Assessment & Reporting Technique (CHART); Impacton Participation and Autonomy Questionnaire (IPAQ); Physical ActivityRecall Assessment for People with Spinal Cord injury (PARA-SCI);Physical Activity Scale for Individuals with Physical Disabilities(PASIPD); and Reintegration to Normal Living (RNL) Index.

For evaluating Lower Limb & Walking, useful tests include: 10 MeterWalking Test (10 MWT); 6-Minute Walk Test (6MWT); Berg Balance Scale(BBS); Clinical Outcome Variables Scale (COVS); Functional Standing Test(FST); Spinal Cord Injury Functional Ambulation Inventory (SCI-FAI);Timed Up and Go Test (TUG); and Walking Index for Spinal Cord Injury(WISC1) and WISCI II.

For evaluating Mental Health, useful tests include: Beck DepressionInventory (BDI); Brief Symptom Inventory (BSI); CAGE Questionnaire;Center for Epidemiological Studies Depression Scale (CES-D andCES-D-10); Depression Anxiety Stress Scale-21 (DASS-21); FatigueSeverity Scale (FSS); Hospital Anxiety and Depression Scale (HADS);Patient Health Questionnaire-9 (PHQ-9); Scaled General HealthQuestionnaire-28 (GHQ-28); Symptom Checklist-90-Revised (SCL-90-R); andZung Self-Rating Depression Scale (SDS).

For evaluating Neurological Impairment and Autonomic Dysfunction, usefultests include: American Spinal Injury Association Impairment Scale(AIS): International Standards for Neurological Classification of SpinalCord Injury; and Surface Electromyography (sEMG).

Other useful evaluation systems for Affected Physiological Systemsinclude: Exercise Self-Efficacy Scale (ESES); Moorong Self-EfficacyScale (MSES); Spinal Cord Injury Secondary Conditions Scale (SCI-SCS);Spinal Cord Lesion Coping Strategies Questionnaire (SCL CSQ); SpinalCord Lesion Emotional Wellbeing Questionnaire (SCL EWQ); and WingateAnaerobic Testing (WAnT).

For assessing Pain, useful tests include: Brief Pain Inventory (BPI);Classification System for Chronic Pain in SCI; Donovan SCI PainClassification System; Multidimensional Pain Inventory (MPI)—SCIversion; Multidimensional Pain Readiness to Change Questionnaire(MPRCQ2); Quantitative Sensory Testing (QST); Tunk's ClassificationScheme; and Wheelchair Users Shoulder Pain Index (WUSPI).

For evaluating Quality of Life and Health Status, useful tests include:Incontinence Quality of Life Questionnaire (I-QOL); Life SatisfactionQuestionnaire (LISAT-9, LISAT-11); Quality of Life Index (QLI)—SCIVersion; Quality of Life Profile for Adults with Physical Disabilities(QOLP-PD); Quality of Well Being (QWB) and Quality of WellBeing-Self-Administered (QWB-SA); Qualiveen; Satisfaction with LifeScale (SWLS, Deiner Scale); Short Form 36 (SF-36); Sickness ImpactProfile 68 (SIP 68); and World Health Organization Quality of Life-BREF(WHOQOL-BREF).

For evaluating Self Care & Daily Living, useful tests include:Appraisals of DisAbility: Primary and Secondary Scale (ADAPSS); BarthelIndex (BT); Frenchay Activities Index (FAT); Functional IndependenceMeasure (FIM); Functional Independence Measure Self-Report (FIM-SR);Klein-Bell Activities of Daily Living Scale (K-B Scale); LawtonInstrumental Activities of Daily Living scale (IADL); Quadriplegia Indexof Function (QIF); Quadriplegia Index of Function Modified(QIF-Modified); Quadriplegia Index of Function-Short Form (QIF-SF);Rivermead Mobility Index (RMI); Self Care Assessment Tool (SCAT); SelfReported Functional Measure (SRFM); Spinal Cord Independence Measure(SCIM); and Spinal Cord Injury Lifestyle Scale (SCILS).

For Sexuality and Reproduction, useful tests include: Emotional Qualityof the Relationship Scale (EQR); Knowledge, Comfort, Approach andAttitude towards Sexuality Scale (KCAASS); Sexual Attitude andInformation Questionnaire (SAIQ); Sexual Behaviour Scale (SBS); SexualInterest and Satisfaction Scale (SIS); Sexual Interest, Activity an; andSatisfaction (SIAS)/Sexual Activity and Satisfaction (SAS) Scales.

For evaluating Skin Health, useful tests include: Abruzzese Scale;Braden Scale; Gosnell Measure; Norton Measure; Skin Management NeedsAssessment Checklist (SMNAC); Spinal Cord Injury Pressure UlcerScale-Acute (SCIPUS-A); Spinal Cord Injury Pressure Ulcer Scale (SCIPUS)Measure; Stirling's Pressure Ulcer Severity Scale; and. Waterlow Scale.

For evaluating Spasticity, useful tests include: Ashworth and ModifiedAshworth Scale (MAS); Pendulum Test (Wartenberg); Penn Spasm FrequencyScale (PSFS); Spinal Cord Assessment Tool for Spastic Reflexes (SCATS);Spinal Cord Injury Spasticit; and Evaluation Tool (SCI-SET).

For evaluating Upper Limb Functionality, useful tests include: Box andBlock Test (BBT); Capabilities of Upper Extremity Instrument (CUE);Graded Redefined Assessment of Strength, Sensibility and Prehension(GRASSY); Grasp and Release Test (GRT); Hand-Held Myometer; Jebsen HandFunction Test (JHFT); Modified Functional Reach Test (mFRT); Six-MinuteArm Test (6-MAT); Sollerman Hand Function Test; Tetraplegia HandActivity Questionnaire (THAQ); and Van Licshout Test Short Version(VLT-SV).

And, for evaluating Wheeled Mobility, useful tests include: 4 FunctionalTests for Persons who Self-Propel a Manual Wheelchair (4FTPSMW); TimedMotor Test (TMT); Tool for assessing mobility in wheelchair-dependentparaplegics; Wheelchair Circuit (WC); and Wheelchair Skills Test (WST).

Based on the effects of myostatin on muscle mass and metabolism, amyostatin inhibitor, e.g., an anti-myostatin antibody or antigen-bindingportion thereof, can potentiate a number of long-term healthconsequences (which may be measured by one or more standardizedtests/tools such as those listed above) which affect those living withSCI, and would cause clinically meaningful benefits to patients at thetime of injury and/or in chronic conditions. Indeed, the presentinventors surprisingly discovered that specific inhibition of myostatinactivation by a myostatin inhibitor, e.g., an anti-pro/latent myostatinantibody had a positive impact on muscle function in the subjects,including in muscles below the injury or lesion. Specifically,administration of the myostatin inhibitor, e.g., anti-pro/latentmyostatin antibody, to a partial denervation animal model not onlyprevented muscle atrophy and increased muscle mass in the injuredsubjects, but also enhanced the function of the injured muscle, as wellas prevented metabolic dysregulation associated with neuron injuriesand, thus, improving the overall metabolic health of the subjects whichmay provide significant long term benefits.

Whilst myostatin inhibition is effective in treating muscle atrophy andmetabolic dysfunction caused by partial/incomplete SCI (such as severecontusion SCI) as described above, where the function of innervatingmotor neurons is at least partially intact, the inventors of the presentdisclosure further contemplate the use of a myostatin inhibitor, such asthose described herein, in the treatment of complete SCI (e.g., completetransection), used in conjunction with a nerve stimulator.

Previously published work indicated that myostatin inhibition wasineffective in ameliorating complete transection injuries of the spinalcord or sciatic nerve. For example, it has been reported thatprophylactic administration of a soluble ActRIIB ligand trap showed notherapeutic effect on muscle atrophy or bone loss in sublesional hindlimbs in a complete transection model of SCI in mice (Graham 2015). Thesame study still showed increased mass of supralesional muscle,suggesting that myostatin inhibition is ineffective in the context ofdenervated muscle. In support of this, a separate study showed thatprophylactic administration of soluble ActRIIB ligand trap failed toprevent muscle atrophy following complete transection of the sciaticnerve (MacDonald 2014).

Nevertheless, based on Applicant's previous recognition thateffectiveness of myostatin inhibition at least in part depends onneuronal signaling from the innervating motor neurons (see, for example,PCT/US2017/037332), it is contemplated that myostatin inhibitor therapy,in conjunction with neuronal stimulation, may enhance therapeuticeffects in complete SCI. Studies in rats and human patients withcomplete transection SCI suggest that a therapeutic neuronal stimulationregimen may protect against sublesional muscle atrophy, the conversionfrom slow twitch to fast fatigable muscle, and bone loss, whileincreasing muscle strength and decreasing blood glucose and insulincompared to control (Wu 2013; Adams 2011; Shields 2006; Griffin 2007).Thus, contrary to the general consensus in the art that myostatininhibition appears ineffective in treating complete transection nerveinjuries, it is contemplated herein to use a myostatin inhibitor,preferably an inhibitor of myostatin activation, in the treatment ofpatients inflicted with complete SCI to enhance clinical benefits ofnerve stimulation.

In some embodiments, suitable neuronal stimulation comprises electricalstimulation, such as functional electrical stimulation or neuromuscularelectrical stimulation. In some embodiments, one or more agents thatsimulate nerve stimulation or effects thereof in vivo may be employed.In some embodiments, such agents cause depolarization at theneuromuscular junction, such as voltage-sensitive sodium channelagonists. In some embodiments, such agents cause elevated calciumconcentrations in the target muscle to mimic membrane potentiation. Insome embodiments, such agents potentiate neurotransmission. In someembodiments, such agents are neurotransmitter agonists, such asacetylcholine or derivative thereof. In some embodiments, such agentsactivate postsynaptic receptors on the target muscle, e.g.,acetylcholine receptors. In some embodiments, such agents inducephosphorylation of postsynaptic component(s) so as to mimicneurotransmission at the neuromuscular junction. In some embodiments,such agents regulate ECM assembly that promote postsynaptic function.For example, the agents may facilitate interactions of extracellularcomponents such as synaptic integrins, laminins, collagens, as well astheir receptors on the target muscle, such as agrin and MuSK.

Such combination therapy may be effective to prevent or amelioratemuscle atrophy, bone loss, and/or metabolic dysrcgulation, in patientssuffering from severe SCI, such as injuries involving complete or almostcomplete transection SCI.

Spinal Muscular Atrophy (SMA)

Myostatin inhibition has been shown to be an effective approach toenhance motor function in SMA, which is a genetic disease associatedwith impaired neuromuscular signaling due to mutations in the Smn1 gene.This concept is captured in more detail in, for example,PCT/US2017/037332 and PCT/US2017/012606. The present disclosure expandsthis notion and encompasses the recognition that clinical benefits ofmyostatin inhibition in SMA may further include prevention oramelioration of bone loss or fracture. In some embodiments, subjectswith SMA who receives myostatin inhibitor therapy, such as thosedescribed herein, may show beneficial clinical effects, as measured byone or more parameters, which include but are not limited to:cross-sectional bone area, cortical thickness, trabecular thickness,trabecular number, and trabecular separation.

Whether or not inhibition of myostatin provides a direct benefit tohone, as opposed to indirect, muscle-driven effects, has been unclear.Data presented herein supports the idea that myostatin inhibition cansurprisingly exert beneficial effects even on non-weight-bearing bone,suggesting that myostatin inhibitors such as those described herein mayat least in part directly target the bone, in addition tomuscle-mediated effects. Thus, the present invention includes the use ofmyostatin inhibitors in the treatment of SMA in an amount effective toprotect against (e.g., prevent or retard) bone loss, and/or reduce thefrequency and/or degree of bone fracture in these patients. In someembodiments, SMA patients include those on neuronal corrector therapy.In some embodiments, SMA patients have not received or are notcandidates for a neuronal corrector therapy. In some embodiments, theSMA patients who are not candidates for a neuronal corrector therapyhave undergone a spinal fusion procedure. In some embodiments, SMApatients have ambulatory SMA (such as Type III). In some embodiments,SMA patients are non-ambulatory (such as Type I and severe forms of TypeII).

C. Other Diseases and Disorders

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to Cachexia. Exemplary diseasesand conditions related to cachexia include, without limitation, cancer,chronic heart failure (CHF), acquired immune deficiency syndrome (AIDS),chronic obstructive pulmonary disease (COPD), and chronic kidney disease(CKD).

A significant fraction of cancer patients suffers from cachexia and/orbone loss/frequent bone fractures. Myostatin inhibitors such as thosedescribed herein may provide clinically beneficial effects in cancerpatients to not only prevent muscle loss but also prevent bone loss andreduce the frequency and/or severity of bone fractures.

In some embodiments, any metabolic bone diseases or diseases associatedwith bone loss (such as cancer) may be treated with a combination of amyostatin inhibitor and at least one other therapy, such as TGFβinhibitor (preferably a TGFβ1 inhibitor) and/or other bone-protectiveagents, e.g., bisphosphonates, calcium, vitamin D, RANKL inhibitors,etc.

Another aspect of the disclosure includes a method of treating a subjecthaving a disease or condition related to rare diseases. Exemplary rarediseases and conditions include, without limitation, osteogenesisimperfecta, sporadic inclusion body myositis, and acute lymphoblasticleukemia.

D. Weight Loss

The present invention further provides methods for promoting robustweight loss in both healthy subjects, e.g., bodybuilders, or in subjectshaving metabolic diseases, such as obesity, e.g., diet-induced obesity,metabolic syndrome, NASH/NAFLD, and/or diabetes. As compared to dietingalone (e.g., dieting by caloric restriction, low-carbohydrate diet,ketogenic diet, vegan diet, etc.), where weight loss occurs in both fatstores and muscle during dieting, administration of a myostatininhibitor disclosed herein in combination with a diet leads to weightloss in fat stores, while sparing the muscle. Specifically,administration of a myostatin inhibitor in combination with a dietresults in more robust weight loss due to the maintenance of a highermetabolic rate; improved cardiometabolic benefits (such as lipidprofile, glucose metabolism, cardiovascular risk, etc.); and higherreduction in visceral fat and other deleterious fat levels as comparedto dieting, alone. Additionally, administration of a myostatin inhibitorprevents or reduces muscle atrophy and/or bone loss which can occurconcomitantly with a diet, e.g., a caloric restriction diet, alow-carbohydrate diet, a ketogenic diet, etc. Overall, administration ofa myostatin inhibitor, e.g., an antibody or antigen-binding portionthereof, in combination with a diet increases the ratio of muscle to fatin the subject.

In subjects with metabolic diseases, e.g., obesity, metabolic syndrome,NASH/NAFLD, and/or diabetes, the combination of a myostatin inhibitorwith a moderate diet enables the same metabolic benefits as a moreaggressive diet, alone. A more moderate diet, e.g., caloric restrictiondiet, provides for better patient compliance and better long-termoutcomes since subjects do not have to adhere to austere, aggressivediets, e.g., aggressive caloric restriction diets.

Such treatments are particularly useful for subjects who havelimitations on physical activity, e.g., subjects having an orthopedicinjury, spinal cord injury, musculoskeletal disease, a pulmonarydisorder, a cardiac disorder, a neurologic disorder, severe obesity,etc. In such subjects, administration of a myostatin inhibitor incombination with a diet, e.g., a caloric restriction diet, preventsmuscle atrophy and/or bone loss which are more prominent in thesesubjects due to their limitations on physical activity. In someembodiments, such a treatment enables subjects with limitations onphysical activity to undergo a more robust diet, e.g., caloricrestriction diet, because they are no longer limited by concernsregarding muscle loss or bone loss due to the administration of themyostatin inhibitor.

In some embodiments, the subject is on a diet, e.g., a caloricrestriction regimen, but is not on an exercise regimen. In someembodiments, the subject is on a dict, e.g., a caloric restrictionregimen, and an exercise regimen.

Kits

The present disclosure also provides kits for use in alleviatingdiseases/disorders associated with myopathy. Such kits can include oneor more containers comprising a myostatin inhibitor, e.g., ananti-pro/latent-myostatin antibody, or antigen binding fragment thereof,e.g., any of those described herein.

In some embodiments, the kit can comprise instructions for use inaccordance with any of the methods described herein. The includedinstructions can comprise a description of administration of themyostatin inhibitor, e.g., anti-pro/latent-myostatin antibody, orantigen binding fragment thereof, to treat, delay the onset, oralleviate a target disease as those described herein. The kit mayfurther comprise a description of selecting an individual suitable fortreatment based on identifying whether that individual has the targetdisease. In still other embodiments, the instructions comprise adescription of administering an antibody to an individual at risk of thetarget disease.

The instructions relating to the use of a myostatin inhibitor, e.g., ananti-pro/latent-myostatin antibody, or antigen binding fragment thereof,generally include information as to dosage, dosing schedule, and routeof administration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the disclosure are typicallywritten instructions on a label or package insert (e.g., a paper sheetincluded in the kit), but machine-readable instructions (e.g.,instructions carried on a magnetic or optical storage disk) are alsoacceptable.

The label or package insert indicates that the composition is used fortreating, delaying the onset and/or alleviating a disease or disorderassociated with myopathy. Instructions may be provided for practicingany of the methods described herein.

The kits of this disclosure are in suitable packaging. Suitablepackaging includes, but is not limited to, vials, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.Also contemplated are packages for use in combination with a specificdevice, such as an inhaler, nasal administration device (e.g., anatomizer) or an infusion device such as a minipump. A kit may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The container may also have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is an anti-pro/latent-myostatinantibody, or antigen binding fragment thereof, as those describedherein.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. In someembodiments, the disclosure provides articles of manufacture comprisingcontents of the kits described above.

Assays for Detecting Pro/Latent-Myostatin

In some embodiments, methods and compositions provided herein relate toa method for detecting pro/latent-myostatin in a sample obtained from asubject. As used herein, a “subject” refers to an individual organism,for example, an individual mammal. In some embodiments, the subject is ahuman. In some embodiments, the subject is a non-human mammal. In someembodiments, the subject is a non-human primate. In some embodiments,the subject is a rodent. In some embodiments, the subject is a sheep, agoat, a cattle, a cat, or a dog. In some embodiments, the subject is avertebrate, an amphibian, a reptile, a fish, an insect, a fly, or anematode. In some embodiments, the subject is a research animal. In someembodiments, the subject is genetically engineered, e.g., a geneticallyengineered non-human subject. The subject may be of either sex and atany stage of development. In some embodiments, the subject is a patientor a healthy volunteer. In some embodiments, the subject is a “healthysubject” (e.g., who is not at risk of developing a muscle condition,such as muscle atrophy, but may nevertheless benefit from increasedmuscle mass and/or function). In some embodiments, the subject has or atrisk of developing muscle atrophy or weakness. In some embodiments, thesubject has or at risk of developing muscle atrophy or weakness and willbenefit from increased muscle mass and/or function.

In some embodiments, a method for detecting a pro/latent-myostatin in asample obtained from a subject involves (a) contacting the sample withthe anti-pro/latent-myostatin antibody, or antigen binding fragmentthereof, under conditions suitable for binding of the antibody to theantigen, if the antigen is present in the sample, thereby formingbinding complexes; and (b) determining the level of the antibody orantigen binding fragment bound to the antigen (e.g., determining thelevel of the binding complexes).

As used herein a binding complex refers to a biomolecular complex ofantibody (including antigen binding fragments) bound to antigen (e.g.,pro/latent-myostatin protein). Binding complexes may comprise antibodieswith a single specificity or two or more antibodies or antigen bindingfragments with different specificities. In one embodiment, a bindingcomplex comprises two or more antibodies recognizing different antigenicsites on the same antigen. In some instances, an antibody may be boundto an antigen, having bound to it other biomolecules such as RNA, DNA,polysaccharides or proteins. In one embodiment, a binding complexcomprises two or more antibodies recognizing different antigens. In someembodiments, an antibody in a binding complex (e.g., an immobilizedantibody bound to antigen), may itself by bound, as an antigen, to anantibody (e.g., a detectably labeled antibody). Thus, binding complexesmay, in some instances, comprise multiple antigens and multipleantibodies or antigen binding fragments.

Antigens present in binding complexes may or may not be in their nativein situ conformation. In some embodiments, a binding complex is formedbetween an antibody and a purified protein antigen, or isolated proteinscomprising antigen, in which the antigen is not in its native in situconformation. In some embodiments, a binding complex is formed betweenan antibody and a purified protein antigen, in which the antigen is notin its native in situ conformation and is immobilized on solid support(e.g., a PVDF membrane). In some embodiments, a binding complex isformed with an antibody and, for example, a cell surface protein that ispresent in situ in a native confirmation (e.g., on the surface of acell).

Antibodies in binding complexes may or may not be detectably labeled. Insome embodiments, binding complexes comprise detectably labeledantibodies and non-labeled antibodies. In some embodiments, bindingcomplexes comprise detectably labeled antigen. In some embodiments,antibodies, in binding complexes, are immobilized to one or more solidsupports. In some embodiments, antigens, in binding complexes, areimmobilized to one or more solid supports. Exemplary solid supports aredisclosed herein and will be apparent to one of ordinary skill in theart. The foregoing examples of binding complexes are not intended to belimiting. Other examples of binding complexes will be apparent to one orordinary skill in the art.

In any of the detection, diagnosis, and monitoring methods, theantibody, (including antigen binding fragments) or antigen may beconjugated to a solid support surface, either directly or indirectly.Methods for conjugation to solid supports are standard and can beaccomplished via covalent and non-covalent interactions. Non-limitingexamples of conjugation methods include: adsorption, cross-linking,protein A/G—antibody interactions, and streptavidin-biotin interactions.Other methods of conjugation will be readily apparent to one of ordinaryskill in the art.

In some aspects, detection, diagnosis, and monitoring methods includecomparing the level of the antibody (including antigen bindingfragments) bound to the antigen (e.g., pro/latent-myostatin) to one ormore reference standards. The reference standard may be, for example,the level of a corresponding pro/latent-myostatin in a subject that doesor does not have a pro/latent-myostatin. In one embodiment, thereference standard is the level of pro/latent-myostatin detected in asample that does not contain pro/latent-myostatin (e.g., a backgroundlevel). Alternatively, a background level can be determined from asample that contains a particular pro/latent-myostatin, by contactingthe sample with non-specific antibodies (e.g., antibodies obtained fromnon-immune serum). Then again, the reference standard may be the levelof pro/latent-myostatin detected in a sample that does containpro/latent-myostatin (e.g., a positive control). In some cases, thereference standard may be a series of levels associated with varyingconcentrations of pro/latent-myostatin in a sample and useful forquantifying the concentration of pro/latent-myostatin in the testsample. The foregoing examples of reference standards are not limitingand other suitable reference standard will be readily apparent to one ofordinary skill in the art. In some embodiments, the level of theantibody bound to pro/latent-Myostatin is compared to the level ofmature myostatin. In some instances, the level of pro/latent myostatinis compared to mature myostatin to determine the ratio of inactive toactive myostatin in the sample.

The level of pro/latent-myostatin may be measured, as provided herein,from a biological sample. A biological sample refers to any biologicalmaterial which may be obtained from a subject or cell. For example, abiological sample may be whole blood, plasma, serum, saliva,cerebrospinal fluid, urine, cells (or cell lysate) or tissue (e.g.,normal tissue or tumor tissue). In some embodiments, a biological sampleis a fluid sample. In some embodiments, a biological sample is a solidtissue sample. For example, a tissue sample may include, withoutlimitation skeletal muscle, cardiac muscle, adipose tissue as well astissue from other organs. In some embodiments, a biological sample is abiopsy sample. In some embodiments, a solid tissue sample may be madeinto a fluid sample using routine methods in the art.

A biological sample may also include one or more cells of a cell line.In some embodiments, a cell line includes human cells, primate cells(e.g., vero cells), rat cells (e.g., GH3 cells, OC23 cells) or mousecells (e.g., MC3T3 cells). There are a variety of human cell lines,including, without limitation, human embryonic kidney (HEK) cells, HeLacells, cancer cells from the National Cancer Institute's 60 cancer celllines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer)cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breast cancer) cells,PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acutemyeloid leukemia) cells, U87 (glioblastoma) cells, SHSY5Y humanneuroblastoma cells (cloned from a myeloma) and Saos-2 (bone cancer)cells.

A further embodiment relates to a method for monitoring a disease, acondition, or any treatment thereof (e.g., myopathy or myopathytreatment) in a subject having, or at risk of having, the disease orcondition comprising: (a) obtaining a biological sample from thesubject, (b) determining the level of a pro/latent-myostatin in thebiological sample using an antibody that detects pro/latent-myostatin,and (c) repeating steps (a) and (b) on one or more occasions. Myostatinhas been used as a biomarker for muscle atrophy, however, the currentlyavailable commercial methods and reagents (e.g., antibodies used inELISAs and Western Blots) are either not specific for myostatin, detectonly mature myostatin or do not detect myostatin at all. Thus, providedherein are methods and reagents (e.g., antibodies) for detectingpro/latent-myostatin in the context of diseases and/or conditions (e.g.,muscle atrophy) for diagnostic purposes. As one example, the level ofpro/latent-myostatin may be measured in a subject, or biological sampletherefrom, to detect or monitor the progression of a disease orcondition. As another example, the level of pro/latent-myostatin may bemeasured in a subject, or biological sample therefrom, to monitor theresponse to a treatment for a disease or condition. It should beappreciated that the level of pro/latent-myostatin may be monitored overany suitable period of time, which may differ depending on the diseaseor condition, the subject has or any treatment regimen that the subjectmay be subject to.

Another embodiment relates to a diagnostic composition comprising anyone of the above described antibodies, antigen binding fragments,polynucleotides, vectors or cells and optionally suitable means fordetection. The antibodies are, for example, suited for use inimmunoassays in which they can be utilized in liquid phase or bound to asolid phasc carrier. Examples of immunoassays which can utilize theantibody are competitive and non-competitive immunoassays in either adirect or indirect format. Examples of such immunoassays are the EnzymeLinked Immunoassay (ELISA), radioimmunoassay (RIA), the sandwich(immunometric assay), flow cytometry, the western blot assay,immunoprecipitation assays, immunohistochemistry, immuno-microscopy,lateral flow immuno-chromatographic assays, and proteomics arrays. Theantigens and antibodies can be bound to many different solid supports(e.g., carriers, membrane, columns, proteomics array, etc.). Examples ofsolid support materials include glass, polystyrene, polyvinyl chloride,polyvinylidene difluoride, polypropylene, polyethylene, polycarbonate,dextran, nylon, amyloses, natural and modified celluloses, such asnitrocellulose, polyacrylamides, agaroses, and magnetite. The nature ofthe support can be either fixed or suspended in a solution (e.g.,beads).

By a further embodiment, antibodies (including antigen bindingfragments) provided herein may also be used in a method for evaluatingpro/latent-myostatin expression in a subject by obtaining a biologicalsample from the subject which may be a tissue sample, a blood sample orany other appropriate body fluid sample. The procedure may comprisecontacting the blood sample (whole blood, serum, plasma), a tissuesample, or protein sample isolated therefrom, with an antibody, underconditions enabling the formation of binding complexes between antibodyand antigen. The level of such binding complexes may then be determinedby any suitable method. In some embodiments, the biological sample iscontacted with the antibody under conditions suitable for binding of theantibody to a pro/latent-myostatin protein, if the antigen is present inthe sample, and formation of binding complexes consisting of antibody,bound to the antigen. This contacting step is typically performed in areaction chamber, such as a tube, plate well, membrane bath, cellculture dish, microscope slide, and the like. In some embodiments, anantibody is immobilized on a solid support. In some embodiments, theantigen is immobilized on a solid support. In some embodiments, thesolid support is the surface of the reaction chamber. In someembodiments, the solid support is of a polymeric membrane (e.g.,nitrocellulose strip, Polyvinylidene Difluoride (PVDF) membrane, etc.).Other appropriate solid supports may be used.

In some embodiments, an antibody is immobilized on the solid supportprior to contacting with the antigen. In other embodiments,immobilization of the antibody is performed after formation of bindingcomplexes. In still other embodiments, antigen is immobilized on a solidsupport prior to formation of binding complexes. A detection reagent isadded to the reaction chamber to detect immobilized binding complexes.In some embodiments, the detection reagent comprises a detectablylabeled secondary antibody directed against the antigen. In someembodiments, the primary antibody is itself detectable labeled, and isthereby the detection reagent.

In one aspect, detection methods comprise the steps of immobilizingantibodies to a solid support; applying a sample (e.g., a biologicalsample or isolated protein sample) to the solid support under conditionsthat permit binding of antigen to the antibodies, if present in thesample; removing the excess sample from the solid support; applyingdetectably labeled antibodies under conditions that permit binding ofthe detectably labeled antibodies to the antigen-bound immobilizedantibodies; washing the solid support and assaying for the presence oflabel on the solid support.

In some embodiments, the antigen is immobilized on the solid support,such as a PVDF membrane, prior to contacting with the antibody in areaction chamber (e.g., a membrane bath). A detection reagent is addedto the reaction chamber to detect immobilized binding complexes. In someembodiments, the detection reagent comprises a detectably labeledsecondary antibody directed against the antigen. In some embodiments,the detection reagent comprises a detectably labeled secondary antibodydirected against the primary antibody. As disclosed herein, thedetectable label may be, for example, a radioisotope, a fluorophore, aluminescent molecule, an enzyme, a biotin-moiety, an epitope tag, or adye molecule. In some embodiments, the primary antibody is itselfdetectable labeled, and is thereby the detection reagent. Suitabledetectable labels are described herein, and will be readily apparent toone of ordinary skill in the art.

Accordingly, diagnostic kits, suitable for home or clinical use (pointof care service), are provided that comprise (a) detectably labeledand/or non-labeled antibodies, as antigen binding reagents (e.g.,pro/latent-myostatin binding reagents); (b) a detection reagent; and,optionally, (c) complete instructions for using the reagents to detectantigens in a sample. In some embodiments, the diagnostic kit includesthe antibody, and/or pro/latent-myostatin immobilized on a solidsupport. Any of the solid supports described herein are suitable forincorporation in the diagnostic kits. In a preferred embodiment, thesolid support is the surface of a reaction chamber of a plate well.Typically, the plate well is in a multi-well plate having a number ofwells selected from: 6, 12, 24, 96, 384, and 1536, but it is not solimited. In other embodiments, the diagnostic kits provide a detectablylabeled antibody. Diagnostic kits are not limited to these embodimentsand other variations in kit composition will be readily apparent to oneof ordinary skill in the art.

The present invention is further illustrated by the following examples,which are not intended to be limiting in any way. The entire contents ofall references, patents and published patent applications citedthroughout this application, as well as the Figures and SequenceListing, are hereby incorporated herein by reference.

EXAMPLES Example 1. Effect of Anti-Myostatin Antibody Treatment onSpinal Cord Injury in Mice

Spinal Cord Injury and Test Article Treatment and Study Measures

The effect of mu-Ab1 on spinal cord injury in mice was studied in amouse severe contusion model. Adult female C57BL/6 mice (8 weeks old)were randomized to four test groups. Mice were anesthetized byintraperitoneal (i.p.) injection using a ketamine (100 mg/kg) andxylazine (10 mg/kg) cocktail, then subjected to a laminectomy betweenthoracic vertebrae T8 and T10 to expose the dorsal surface of the spinalcord. To induce spinal cord injury, the spinal cord at T9 was placeddirectly under the vertical shaft of the Infinte Horizon Impactor(IH-0400 impactor, Precision Systems Instrumentation, LLC, Virginia,USA), followed by slowly lowering of the shaft until the response peakon the force transducer reached the predetermined force level (65kDyne). The control group was subjected to laminectomy (only) at the T9level (sham-operation) Immediately following injury animals wereadministered by i.p. injection with test articles—either vehicle (20 mMCitrate and 150 mNI Sodium Chloride, pH 5.5), IgG (40 mg/Kg), or GDF8(Mu-Ab1, 40 mg/Kg). Follow-up injection of test articles wasadministered in the same manner 1-week post-SCI. During the two-weekstudy multiple physical and behavior measures were used to assess theeffects of anti-myostatin pharmacotherapy. Physical measures includedtotal body weight, muscle weight, total body composition (lean body mass(LBM), fat mass, and bone mineral density) and total metabolic energyexpenditure determined using indirect calorimetry. Behavioral measureswere also assessed including BMS motor score, rotarod test, andgrip-strength test. Between-group differences were analyzed usingone-way ANOVA, followed by Tukey post hoc comparison (GraphPad, Prism).Data are expressed as mean±SEM. A significance level of p<0.05 wasaccepted as different from control.

Results and Data Analysis

Body Mass, Muscle Mass, and Body Composition

Body mass was measured at the following time-points: 0 (Baseline: priorto survival surgery); 1-week post-surgery; and 2-weeks post-surgery(FIG. 3). There were no significant group differences in mass atbaseline. 1-week following SCI (and treatment), there was a significantreduction in body mass in the SCI-veh (P<0.0001) and SCI-IgG (P<0.0001)groups, compared to sham control. There was no statistical difference inbody mass between the sham control and SCI-GDF8 (Mu-Ab1) (P=0.2805)group, However, the SCI-GDF8 (Mu-Ab1) group mass was significantlygreater than both SCI-veh (P=0.004) and SCI-IgG (P=0.0003) groups.2-weeks post-SCI, body mass in SCI-veh (P=0.0011) and SCI-IgG (P=0.0009)remained significantly lower than sham-control. Body mass in theSCI-GDF8 (Mu-Ab1) group remained significantly greater than SCI-veh(P=0.0152) and SCI-IgG (P=0.0123) groups, but not different compared tosham control (P=0.585).

The data indicate that SCI induced a significant decrease in total bodymass when compared to uninjured mice. GDF8 (Mu-Ab1) as a treatmentsignificantly attenuated loss of body mass observed with SCI, such thatgroup means between uninjured and GDF8 (Mu-Ab1) treated mice arequalitatively similar and statistically non-significant.

At necropsy—2-weeks post-SCI—several muscle tissues (soleus,gastrocnemius, biceps and triceps) were extracted to evaluate the effectof SCI and treatment on wet weight (FIG. 4). The average weight of thesoleus muscle was significantly less in the SCI-veh and SCI-IgG (bothP's <0.0001) groups than the sham control. There was no statisticaldifference in soleus mass between the sham control and SCI-GDF8 (Mu-Ab1)(P=0.3129) group, however, the SCI-GDF8 (Mu-Ab1) group soleus mass wassignificantly greater than both SCI-veh and SCI-IgG (both P's<0.0001).Similarly, the average weight of the gastrocnemius muscle wassignificantly less in the SCI-veh and SCI-IgG (both P's<0.0001) than thesham control. There was no statistical difference in soleus mass betweenthe sham control and SCI-GDF8 (Mu-Ab1) (P=0.3255) group, however theSCI-GDF8 (Mu-Ab1) group soleus mass was significantly greater than bothSCI veh and SCI-IgG (both P's<0.0001).

The average mass of the biceps muscle was also significantly less in theSCI veh (P=0.045) and SCI-IgG (P=0.04) groups when compared to shamcontrol. Group mean trends in biceps mass between the SCI-GDF8 (Mu-Ab1)group were greater than both SCI-veh and SCI-IgG groups. The averagemass of the triceps muscle was also significantly less in the SCI-veh(P=0.007) and SCI-IgG group (P=0.0013) compared to sham control. TheSCI-GDF8 (Mu-Ab1) group triceps mass was significantly greater than bothSCI-veh and SCI-IgG (both P's<0.0001).

The data show that sublesional muscle mass—including the primarilyoxidative soleus muscle, and the primarily glycolytic gastrocncmiusmuscle—was significantly reduced following SCI when compared touninjured mice. GDF8 (Mu-Ab1) treatment surprisingly and significantlyattenuated this muscle loss, where both soleus and gastrocnemius meanmuscle mass was equal to the mass of uninjured mice, suggesting aneffect across muscle phenotype. When examining supralesional musclemass—including biceps and triceps muscles—there was also a significantreduction in mass with SCI compared to uninjured mice. This is likelydue to an overall depression of physiological systems and global loss ofmass with SCI (albeit a greater proportion of muscle mass loss issublesional).

At 2-weeks post-SCI body composition (lean and fat mass) was assessed bydual-energy x-ray absorptiometry (DXA) densitometry (Lunar PIXImus™densitometer (GE Medical-Lunar, Madison, Wis.)) in all experimentalgroups. Total body fat-free (lean) mass was significantly less in theSCI-veh (P=0.0124) and SCI-IgG (P=0.056) groups compared to shamcontrol. The SCI-GDF8 (Mu-Ab1) group total fat-free (lean) mass wassignificantly greater than both the SCI-veh (P=0.0254) and SCI-IgG(P=0.0114) groups (FIG. 5), and group mean trends indicated greater fatmass in both the SCI-veh and SCI-IgG groups when compared to shamcontrol (FIG. 5). The SCI-GDF8 (Mu-Ab1) group mean trend for whole bodyfat mass was also less than both the SCI-veh and SCI-IgG groups, andcomparable to sham control. When examining the average fat-free (lean)mass as a percentage of body mass, there were no discernable differencesbetween groups, suggesting that changes in body mass after SCI, andtreatment effects of GDF8 (Mu-Ab1) were limited to changes in lean bodymass (FIG. 6).

These data show that total—or whole body—fat free (lean) mass wassignificantly reduced following SCI compared to sham control. GDF8(Mu-Ab1) treatment significantly attenuated this loss of fat-free (lean)mass after SCI, where mean fat-free (lean) mass was not different fromuninjured mice, suggesting an effect on global lean tissue. Conversely,total fat mass after SCI appeared to increase compared to sham control,when examining group means. The reduction in sublesional fat-free (lean)mass, and the increase in adiposity (global and regional) is a wellcharacterized feature of chronic SCI pathophysiology.

Intramuscular infiltration of fat/lipid following SCI was histologicallyanalyzed. Fresh frozen gastrocnemius (GN) and soleus (SOL) muscle,harvested two-weeks post-SCI, were sectioned and stained with Oil Red Ofor visualization of neutral lipids.

As the images provided in FIG. 27A show, there was a significantincrease in the area of Oil Red O stain in SCI-IgG and SCI-Mu-Ab1 tissuesamples, as compared to sham in both muscle types. The scale barrepresents 50 micrometers.

The area of Oil Red O stain in the antibody-treated group wassignificantly reduced as compared to SCI-IgG control for both muscletypes. The results are summarized in FIG. 27B (GN) and FIG. 27C (SOL).

Metabolism and Total Energy Expenditure

Indirect calorimetry was performed on mice using a 12-chamberopen-circuit Oxymax system of the Comprehensive Lab Animal MonitoringSystem (CLAMS; Columbus Instruments, Columbus, Ohio, USA). Mice weretransferred to individual metabolic chambers for 3-days prior to (andincluding) the 2-week post-SCI analysis time-point. VO₂ and VCO₂ weremeasured continuously, and using indirect calorimetry, energyexpenditure/hour (kcal/hr) and total energy expenditure/day (TEE) werecalculated (FIG. 7). Group mean trends suggest a decrease in thesemeasures in SCI-veh and SCI-IgG compared to sham control, and that themetabolic decrease in the SCI-veh group versus sham control approachedstatistical significant (P=0.075). Group means trended toward elevationin the SCI-GDF8 (Mu-Ab1) group when compared to the SCI-veh and SCI-IgGgroups. Notably, the metabolic increase in the SCI-GDF8 (Mu-Ab1)compared to the SCI-veh approached statistically significant (P=0.0530),and the direction of the SCI-GDF8 group mean appeared slightly elevatedcompared to sham control. For additional analysis, the SCI groups notreceiving the GDF8 (Mu-Ab1) treatment drug (SCI-veh+SCI-IgG) wascollapsed to add group power to the SCI treatment control. In doing so,it was found that kcal/hr and TEE in the coalesced SCI-treatment controlgroup was lower than the sham control (P=0.0159). There was nostatistical difference in kcal/hr and TEE between the sham control andSCI-GDF8 (Mu-Ab1) (P=0.9764) group, however the SCI-GDF8 (Mu-Ab1) groupkcal/hr and TEE was significantly greater than the coalescedSCI-treatment control (P=0.0106) group.

The results show that metabolism (as energy expenditure) was depressedfollowing SC1, and that GDF8 (Mu-Ab1) treatment maintained restingmetabolism at levels that approximate uninjured controls. These resultsfurther suggest that the biological effect of GDF8 (Mu-Ab1) on leantissue, in particular muscle preserves levels of resting metabolism thatare otherwise reduced following SC1.

Functional Measures

BMS Open Field Locomotor Test

The Basso Mouse Scale (BMS) open field locomotor test (using a 0 to 9rating system) was used to assess recovery of hind-limb locomotorfunction following SCI, including (but not limited to) variables such asfoot placement, weight support, and joint motion. Under blindedconditions, a team of two investigators evaluated the mice over a4-minute time period at baseline, 1 day after SCI/sham, and weeklythereafter. The arena was divided into three zones (wall, inter andcenter) and mouse behavior was recorded over a 5-minute period using ahigh resolution, video camera. The total number of lines crossed, timespent in each zone, and stereotypical behaviors such as grooming andrearing were analyzed and expressed as number of events.

There was a significant reduction in BMS composite score in the SCI-vehand SCI-IgG groups (both P's<0.0001) 1-day post-SCI (FIG. 8). Because ofthe uniformity at this time-point in the SCI-veh and SCI-IgG groups,they were collapsed to provide additional study power at latertime-points (SCI-treatment control). Also, at 1-day post-SCI, there wasa significant reduction in BMS score in the SCI-GDF8 (Mu-Ab1) group(P<0.0001) compared to sham control. 1-week post-SCI, BMS scoresremained significantly reduced in SCI-treatment control (P<0.0001) andSCI-GDF8 (Mu-Ab1) (P=0.0128) groups compared to sham control. However,the BMS score was significantly greater in the SCI-GDF8 (Mu-Ab1)(P=0.0148) group compared to the SCI-treatment control. Similarly, at2-weeks post-SCI, BMS scores remained significantly reduced in both theSCI-treatment control (P<0.001) and SCI-GDF8 (Mu-Ab1) (P=0.0182) groups,but again, the SCI-GDF8 (Mu-Ab1) group had a significantly higher BMSscore than the SCI treatment control (P=0.0143).

Rotarod Test

Motor coordination and balance were tested on the accelerating rotarodcylinder (Rotamex 4/8, Columbus Instruments). The procedure consisted ofa 5-day pre-training (days 1 to 5) followed by the testing (1-week and2-weeks post-SCI/sham). The cylinder rotated at increasing speed andconstant acceleration (from 10 to 60 rpm over 10-minute period). Thetotal time spent on the rod prior to fall was recorded and non-walkingbehaviors, such as passive clinging to the rod, were manually corrected.Each trial consisted of an average of 4 sessions.

Using rotorod time trials as a proxy measure of motor coordination andbalance this study showed that 1-week post SCI, there was a significantdecrease in average rotarod time in the SCI-veh, SCI-IgG, and SCI-GDF8groups (all P's<0.0001) compared to sham control (FIG. 9). The SCI-GDF8(Mu-Ab1) group mean was greater than both the SCI-veh and SCI-IgG groups(P=0.118). Similarly, 2-weeks post SCI, a significant decrease persistedin average rotarod time in the SCI-veh, SCI-IgG, and SCI-GDF8 (Mu-Ab1)groups (all P's<0.0001) compared to sham control. Again, although therewas no statistical difference between any of the SCI groups, theSCI-GDF8 (Mu-Ab1) group mean was greater than both the SCI-vch andSCI-IgG groups (P=0.1708).

Grip Strength Test

All animals from the sham and SCI groups underwent analysis of hindlimbpeak force (muscle strength) using the grip-strength test. Hind-limbgrip strength was assessed using a digital force gauge (Chatillon DFIS2,Ametek), which generates a measure of neuromuscular function as maximalmuscle strength—with the unit of force measured in grams. The testconsisted of a baseline assessment prior to surgery, followed by a testday at 1-week and 2-weeks post-surgery. Force values were the calculatedaverage of 5-trials.

One week post-SCI, there was a significant decrease in grip strength inthe SCI-veh, SCI-IgG, and SCI-GDF8 (Mu-Ab1) groups (all P's<0.0001)compared to sham control (FIG. 10). The SCI-GDF8 (Mu-Ab1) group gripstrength was significantly greater than both the SCI-veh (P=0.0006) andSCI-IgG (P=0.0003), although the latter two groups were not differentfrom each other. Two weeks post-SCI, there was a significant decrease ingrip strength in the SCI-veh, SCI-IgG, and SCI-GDF8 (Mu-Ab1) groups (allP's<0.0001) compared to sham control. Grip strength for the SCI-GDF8(Mu-Ab1) group was significantly greater than the SCI-veh group(P=0.0124), although not statistically different from the SCI-IgG group(however the group mean trended to greater strength; P=0.1856).

The results indicate that SCI causes a drastic reduction in hind-limblocomotor function (BMS), translating to marked reduction in motorcoordination and balance (rotarod), as well as muscle strength (gripstrength). GDF8 (Mu-Ab1) treatment prevented this change, as thecomposite BMS score for GDF8 was significantly greater than the otherinjury groups Animals treated with GDF8(Mu-Ab1) also had higher motorcoordination and balance as assessed by the rotarod time trials.

In conclusion, these data demonstrated a profound effect of GDF8(Mu-Ab1) treatment on the anthropometric, physiological, and functionaloutcome measures of mice with SCI. SCI-induced reduction in body massand sublesional muscle mass were attenuated with GDF8 (Mu-Ab I), andmetabolic abnormalities associated with SCI—related to body compositionand energy expenditure—were less pronounced following GDF8 (Mu-Ab1)treatment. Further, the effects of GDF8 (Mu-Ab1) treatment translate tolocomotor and functional benefits when compared to the non-treated SCIcondition.

Example 2. Effects of Treatment with Ab2 on Lean Mass, Muscle Weight,and Serum Myostatin in Healthy Cynomolgus Monkeys

Effects of treatment with Ab2 on change in lean mass were evaluated inhealthy Cynomolgus monkeys (n=6 per treatment group). Healthy maleCynomolgus monkeys (avg age: 34 months at start of study) were dosed byintravenous injection once weekly for 8 weeks at three different doselevels of Ab2 (3 mg/kg, 10 mg/kg, and 30 mg/kg) with a 4-week recoveryphase. Control animals were administered vehicle control (20 mM Citrateand 150 mM Sodium Chloride USP, pH 5.5). Lean mass was measured by DualEnergy X-Ray Absorptiometry (DEXA) at baseline and at intervalsthroughout the 12 week study (FIGS. 11A-11D). Treatment with Ab2resulted in a 5-9% increase in the limb lean mass of Ab2-treated monkeyscompared to vehicle control (FIGS. 11A-11D and FIG. 13). Effects oftreatment with Ab2 on tissue weights from the biceps brachii andgastrocnemius muscles of healthy Cynomolgus monkeys were also measuredat week 12 (FIGS. 12A-12B). Significant effects of Ab2 treatment wereapparent in the weights of these muscles (FIGS. 12A-12B and FIG. 13).The gastrocnemius and biceps brachii muscles, which are rich in fasttwitch fibers, were substantially larger by as much as 25% inAb2-treated animals compared to the vehicle control (FIG. 13).Therefore, Ab2 treatment had a notable effect on muscle growth. Further,Ab2 treatment had a particularly robust effect on fast twitch-richmuscle fibers.

Throughout this study, serum samples for analysis of serum Myostatinlevels were collected on study days 2, 4, 8, 15, 22, 29, 36, 43, 64, and85 as indicated in FIGS. 14A-14B. Effects of treatment with Ab2 onlatent Myostatin levels in the serum were also evaluated usingquantitative fluorescent western blotting. Increase in Myostatin levelsin Ab2-treated animals peaked and plateaued between study days 15 and 29and declined by study day 85. Increase in latent Myostatin levels in theserum was seen with all doses of Ab2 with greatest increase seen inanimals treated with 30 mg/kg of Ab2 (FIGS. 14A-14B).

Example 3. Effects of Myostatin and Myostatin Inhibition on GeneExpression

A. Natural History Study in Rats

In order to understand the effect of myostatin on signaling pathwaysafter nerve injury, Quantigene analyses of gastrocnemius muscles andspinal cord from a rat severe contusion injury (SCI) model wereperformed. The study utilized negative control and SCI rats; sampleswere analyzed at six hours, 1 day, 3 days, 5 days, 7 days, and 14 dayspost-injury. Table 4 presents categories of genes that were selected foranalysis, along with the rationale for selection.

TABLE 4 Genes Selected for Analysis Cate- Syno- gory Gene nym RationaleMuscle MSTN negative regulator of muscle targets fbxo32 mafbxupregulated by pSMAD 2/3 trim63 murf1 upregulated by pSMAD 2/3 NRF1upregulated by pgc1a; mitochondria biogenesis NFE2L2 NRF2 upregulated bypgc1a; mitochondria biogenesis Ppargc1a PGC1a mitchondria, metabolism,lipid regulator Slc2a4 glut4 decreased in muscle atrophy; insulininsensitivity Gadd45a upregulated in denervation atrophy, downregulatedwith FES Map3k4 upregulated in denervation atrophy Fndc5 Irisinexpressed by muscle, may affect adipocyte metabolism Ddit4 Redd1upregulated in immobolization and dex induced atrophy IL-6 upregulatedin muscle with SCI; contributes to inflammation; may induce anabolicpathways CNS GDF5 upregulated following denervation; targets lossworsens atrophy Ntf3 neurotrophic Bdnf neurotrophic Gdnf neurotrophicNgf neurotrophic gdf11 closely related to myostatin Lrrc32 garpregulator of TGFβ Lrrc33 regulator of TGFβ, upregulated in mouse spinalcord injury Ltbp1 regulator of TGFβ Ltbp2 regulator of TGFβ Ltbp3regulator of TGFβ, upregulated in mouse spinal cord injury Tgfb1upregulated in mouse spinal cord injury Tgfb2 other tgf beta Tgfb3 othertgf beta rtn4 nogo present in myelin, neurite growth inhibitor lingo-1nogo receptor, upregulated in rat spinal cord 14 dpi bmp7 regulator ofbone Serpine1 tgf beta transcriptional target House HPRT House keeperkeepers GUSB House keeper B2M House keeper PPIB House keeper Polr2aHouse keeper txn2 House keeper rpl19 House keeper ppia House keeper

In the spinal cord, Fbxo32 exhibited decreased expression initiallyfollowing injury, but returned to baseline by the 14-day time point.IL-6, Gdnf, and Pai-1 exhibited increased expression within 6 hours ofinjury, but returned to baseline shortly thereafter. Nfe2L2, Gadd45a,TGFb1, TGFb2, TGFb3, LTBP1, LTBP3, GARP, LRRC33, and BMP7 exhibitedincreased expression following injury; and Ppargc1a, irisin, and Lingo1exhibited decreased expression following injury.

In gastrocnemius, Mstn, Fbxo32, Murf1, Nrf1, Nfe2L2, Gadd45a, GDF5,TGFb2, LTBP1, and Ddit4 exhibited increased expression initiallyfollowing injury, but returned to baseline by the 14 day time point.Glut-4, Irisin, Nogo, and BMP7 exhibited decreased expression followinginjury, but returned to baseline by the 14-day time point. Ppargc1aexhibited decreased expression following injury.

The data indicate that transcriptional changes happen early in the ratSCI model and typically return to baseline by the 14-day time point.

B. Gene Expression in Mouse SCI Model after Treatment with MyostatinInhibitor

In a second study, Quantigene analysis of gastrocnemius from 14 dayspost SCI in a mouse model was performed. The mice were eitheradministered an antibody that inhibits myostatin activation, PBS(vehicle), or IgG. Genes analyzed were Murf-1, Fbxo32, Mstn, Mt2(metalothionein 2), and Ctsl (Cathepsin L) all showed a statisticallysignificant increase in expression after injury. However, mice receivingtreatment with the myostatin inhibitory antibody demonstrated expressionlevels comparable to the sham group (no injury), confirming thattreatment with a myostatin inhibitor prevents upregulation of atrogenes.

C. RNASeq Data from Mice Treated with Myostatin Inhibitor

To broadly profile the changes in muscle brought about by Ab2-mediatedMyostatin inhibition, transcriptional profiling of RNA isolated from thetibialis anterior muscles of drug treated mice was performed. RNA washarvested from muscles tissue collected 3, 7, and 28 days after asingle, 5 mg/kg dose of Ab2. This dose was sufficient to induce a markedand sustained increase in lean mass in treated mice (FIG. 15). RNAharvested from control IgG-treated mice on the day of injection (day 0)was used as a control to measure gene expression at the start of thestudy. For each time point, a minimum of 3 biological replicates wasanalyzed.

After generation of cDNA, transcriptional profiles were assessed by RNAsequencing (RNAseq). Raw sequences were generated by NextGen Sequencing(NGS) followed by alignment to reference sequences. Raw data from thegroups were subsequently analyzed by NOISeq (Tarazona et al., GenomeRes., 2011) to identify differentially expressed genes (DEGs) inAb2-treated groups vs. baseline controls. DEGs for each group werefiltered for statistical significance, then analyzed for coordinateregulation at additional time points (e.g., DEGs upregulated in the day3, day 7, and day 28 datasets). This yielded a total of 138 genes withdifferential expression vs. baseline control at all three timepointsanalyzed. Consistent with a biological response to an inhibitor, themajority of DEGs were downregulated, with only 6 upregulated genes (FIG.16).

RNAseq identified a number of genes previously identified as targets ofMyostatin signaling. Among these are Fbxo32 (Atrogin-1) and Trim63(MuRF). Both of these genes encode ubiquitin ligases that are criticaldrivers of muscle atrophy (Reviewed by Cohen et al., Nat. Drug Discov.,2015) and whose transcription is upregulated by Myostatin signaling.These ligases, as well as one of the genes encoding ubiquitin proteins,polyubiquitin C (Ubc), are downregulated 3- to 10-fold in Ab2-treatedmuscles (FIG. 17), consistent with their previously described inhibitionduring muscle hypertrophy.

The DEG dataset also included genes encoding several muscle-specificstructural proteins, including early upregulation of alpha cardiacmuscle actin (Actc1) (FIG. 18). This form of actin, is associated withearly stages of myogenesis (Tsao et al., Stem Cell Res. Ther., 2013) andhas been previously shown to be upregulated by anti-Myostatin treatment(Latres et al., Skeletal Muscle, 2015). Downregulation of Myogenin(Myog) was observed (FIG. 18), consistent with fusion of myocytes toaugment existing myofibers (Reviewed by Bentzinger et al., Cold SpringHarb. Perspect. Biol., 2012). In addition, several muscle-specificproteins are downregulated by Ab2 treatment, including Myostin LightChain 2 and 4 (My12 and My14) and Troponin C (Tnnc1). Tnnc1 and My12 aremore closely associated with slow twitch skeletal fibers (Amann et al.,Dev Biol, 2014; Lee and Hwang, Gene, 2015), while My14 is associatedwith undifferentiated and fetal muscle (Schiaffino et al., SkeletalMusc., 2015), suggesting some degree of dedifferentiation and/or fibertype switching during anti-Myostatin-driven muscle hypertrophy.

Several investigators have noted an inverse relationship between musclefiber size and muscle respiratory capacity (reviewed by van Wessel etal., Eur. J. Appl. Physiol., 2010), likely reflecting the greateranaerobic demands of larger muscles. Consistent with this, adownregulation of several genes associated with respiratory capacity andmitochondrial synthesis was observed, including PGC-1α, Nor1, UCP-1, andNur77 (FIG. 19), suggesting that anti-Myostatin treatment altersmetabolic function in these hypertrophic muscles.

A large number of DEGs in the dataset reflect changes in adipocytedifferentiation and adipose deposition in Ab2 treated mice. Markers ofdifferentiated adipocytes (Agt, Angptl4, ApoC1) and expression ofadipokines (systemic signaling proteins produced by adipocytes),including adiponectin (Adipoq), Leptin (Lep), Resistin (Res), andhaptoglobin (Hp) are downregulated (FIG. 20). Myostatin inhibition haspreviously been shown to inhibit adipocyte differentiation in vitro(reviewed by Singh et al, Front. Cell Dev. Biol., 2014), and these dataare consistent with those prior results.

Reduced expression of genes associated with increased adipogenesis(Accl, Adipogenin (Adig), Cebpd, Fatty Acid Binding Protein 5 (Fabp5),Fatty Acid Synthase (Fasn), Hormone sensitive lipase E (Lipe), andperilipn 1 and 4 (Plin1 and Plin4) was observed. In addition, we noteupregulation of Sharpl, a negative regulator of adipogenesis (Gulbagciet al., EMBO Rep., 2009). Taken together, these DEGs suggest asignificant decrease in the visceral fat within Ab2 treated muscle.

Myostatin inhibition has previously been shown to alter the phenotype ofadipose tissue, driving white adipose tissue (WAT) towards a brownadipose tissue (BAT) phenotype (reviewed by Singh et al., Singh et al,Front. Cell Dev. Biol., 2014). One of the hallmarks of BAT is theupregulation of PGC-1α and UCP-1, while in our dataset we observe adownregulation of these markers. This may be explained, however, by theoverall reduction in fat within the Ab2-treated muscle such that, evenif browning of the adipose tissue is occurring, we cannot detect itwithin this large geneset.

In addition to the above DEGs, we also noted changes reflectingregulation of the pyruvate dehydrogenase (PDH). PDH is a criticalregulator of the Randle cycle, a biochemical mechanism that regulatescellular usage of glucose vs fatty acids as a primary source (reviewedby Hue and Taegtmeyer, Am J Physiol Endocrinol Metab, 2009). Thecapacity of cells to switch between metabolism of glucose and fattyacids has been termed metabolic flexibility (Storlien et al., PNAS,2004) and has been tightly related to type 2 diabetes, obesity, andmetabolic syndrome (Zhang et al., Nutrition and Metabolism, 2014). PDHactivity is controlled by two regulators: pyruvate dehydrogenase kinase4 (pdk4) and pyruvate dehydrogenase phosphatase (pdpl). Pdk4 inhibitsPDH activity by phosphorylating the protein, while Pdpl activates PDH bydephosphorylation, (FIG. 21). Inactivation of PDH limits conversion ofpyruvate into acetyl-CoA in muscle, and this in turn leads to reducedsynthesis of malonyl-CoA, an important inhibitor of fatty acid oxidation(Foster, J Clin Invest, 2012). Both knockdown and knockout of Pdk4(leading to PDH inactivation) lead to improved glycemic control andglucose tolerance in mice (Tao et al., PLoS One, 2013), suggesting thatPDH is critical to insulin sensitivity.

In Ab2-treated mice, Pdk4 is downregulated and Pdp1 is upregulated,suggesting activation of PDH (FIG. 22). This is further reflected bydownregulation of Acetyl-CoA carpoxylase (Acct), an inhibitor ofmalonyl-CoA production (reviewed by Hue and Taegtmeyer, Am J PhysiolEndocrinol Metab, 2009). Together these data suggest a shift towardsfatty acid metabolism in treated mice, suggesting that at least some ofthe improved glycemic control observed in studies of Myostatininhibition (Dong et al., Int J Obes, 2016) may be a consequence ofdirect effects on muscle rather than on cross talk between muscle andadipose tissue, as previously suggested.

Taken together, data from RNA sequencing of transcripts from Ab2-treatedmice suggest that Myostatin inhibition is a powerful regulator of muscleand adipose metabolism. Furthermore, the data indicating that Myostatinregulates fatty acid oxidation through PDH suggest a potential novel usefor Myostatin inhibitors in increasing metabolic flexibility in patientsexhibiting insulin resistance.

Example 4: Intracellular Versus Secreted proMyostatin

Methods

Immunofluorescence

Tibialis anterior (TA) muscles were fixed in ice cold 4%paraformaldehyde (EMS), PBS for 30 min, incubated overnight in 10%sucrose, PBS at 4° C., then incubated overnight in 20% sucrose, PBS.Muscles were then mounted on cork with tragacanth (Sigma) and frozen inliquid nitrogen cooled isopentane (Sigma) for cryosectioning. 10 μmsections of TA muscle were permeabilized with 0.1% Triton-X 100 (Sigma),PBS for 20 minutes, washed once with 0.05% Triton-X 100, PBS (PBS/T),and then incubated in Mouse IgG blocking reagent (Vector Lab) diluted at1 drop per 1.5 mL PBS/T for 1 h. Sections were washed once with PBS/Tand then incubated in 10% Normal Goat Serum (Sigma), 1% Blocking powder(Perkin Elmer), PBS/T (NGB) for 30 minutes at room temperature. Primaryantibodies (Rabbit anti-laminin, 1:5000, Abeam; Ab10, 50 μg/mL; HuNeg,50 μg/mL,) were diluted in NGB and applied to sections overnight at 4°C. Sections were washed 3 times with PBS/T, and then incubated insecondary antibodies (Alexa Fluor 488 conjugated Goat anti-Rabbit,1:1000, Invitrogen; Alexa Fluor 594 conjugated Goat anti-Human IgG FCY,1:500, Jackson ImmunoResearch) diluted in NGB for 1 h. Sections werethen incubated in 350 nM DAPI (Thermo), PBS/T for 5 minutes, washedtwice with PBS/T, and then mounted with Vectashield (VectorLaboratories). For recombinant protein absorption experiments, 50 μg/mLAb10 was incubated overnight alone or with 10× molar excess of eitherrGDF8 or rGDF11 (both murine) in NGB, and then used as primary antibody.

Microscopy

Fluorescent images were captured with a Leica DM4 B equipped with40×/0.80 Fluotar objective using Leica Application Suite X software.Images were then processed with Fiji (Schindelin, J.; Arganda-Carreras,I. & Frise, E. et al. (2012), “Fiji: an open-source platform forbiological-image analysis”, Nature methods 9(7): 676-682, PMID22743772).

Results

Previous data suggested that the majority of myostatin found in themuscle is stored as pro-myostatin. However, these methods cannotdiscriminate between intracellular and secreted stores of pro-myostatin.To address this, immunofluorescence was performed on cryosectioned TAmuscle from healthy mice using antibody Ab10 that specifically detectspro- and latent myostatin.

Control experiments to test the specificity of anti-pro/latent GDF8antibody, Ab10, are shown in FIGS. 24A-24B and FIGS. 25A-25C. FIGS.24A-24B show cross sections of TA muscle probed with anti-pro/latentGDF8 antibody, Ab10, or a non-specific targeting antibody. Ab10 is shownin FIG. 24A, HuNeg is shown in FIG. 24B, each of the figures arecounterstained with DAPI. The scale bar is 0.01 cm. FIGS. 25A-25C showcross sections of TA muscle probed with anti-pro/latent GDF8 antibody,Ab10, that had been incubated in blocking buffer alone (FIG. 25A),incubated in blocking buffer with 10-fold molar excess a recombinantmouse GDF8 (FIG. 25B), or incubated in blocking buffer with 10-foldmolar excess of recombinant mouse GDF11 (FIG. 25C). FIGS. 26A-26C arecounterstained with DAPI.

Co-staining of anti-pro/latent GDF8 antibody, Ab10, with laminin, anextracellular matrix marker, demonstrated that the majority of myostatinprecursors detected in muscle are in the extracellular space with littlesignal detected intracellularly. FIGS. 23 and 26A-26C show crosssections of TA muscle probed with anti-pro/latent GDF8 antibody, Ab10,and anti-laminin, and counterstained with DAPI. Pro/latent GDF8 andlaminin colocalize in the interstitial space at muscle fiber vertices(arrow), between muscle fibers (arrow head), and around interstitialnuclei (asterisk). Thus, in healthy muscle, pro-myostatin lies dormantin a supracellular space, and Ab10 recognizes the major forms ofmyostatin found in muscle.

Example 5: Effects of Myostatin Inhibition on Bone in an Injury Model

A monoclonal antibody that specifically binds pro/latent forms ofmyostatin and blocks the activation of mature myostatin was evaluatedfor its in vivo effects on bone in a cardiotoxin (CTX)-induced Tibialisanterior (TA) acute injury model in mice. Following a 7-day acclimation,animals (n=19 per group) were treated with weekly doses of 40 mg/kg forfour weeks. The first dose was given pre-injury (one day before CTXtreatment). Doses 2-4 were administered post-injury. Tibia tissues wereharvested for bone microCT scan (5 mice per time point).

Raw microCT images of proximal tibia showed a visibly clear increase intrabecular bone in the antibody-treated mice as compared to control mice(images now shown). Results from quantitative analyses are provided inFIG. 28.

FIG. 28A shows that mice treated with the antibody that inhibitsmyostatin activation increased trabecular bone in proximal tibia.Notably, muscle injury had little impact on trabecular bone; yet, thepro/latent myostatin-targeting drug increased trabecular bone mass.

FIG. 28B provides effects on cortical bone in proximal tibia. Corticalbone effects in the antibody-treated animals was present but lesspronounced than seen in proximal tibia. An increase in the crosssectional area and outer (periosteal) perimeter of the cortical bone inthe antibody-treated animals was seen. It should be noted that corticalbone tends to exhibit smaller changes in bone quality relative totrabecular bone.

Example 6: Effects of Myostatin Inhibition in Murine Model of Mild SMA

To assess the effects of an antibody which inhibits myostatin activationon both muscle function and bone in SMA, a variant of a pharmacologicalmodel of SMA, in which the severity of the disease can be moderatedthrough administration of varying amounts of the small molecule SMN2splice modulator SMN-C1, was used. The foundation of this model is the47 mouse model of severe SMA. This mouse, which lacks the soleendogenous murine SMN gene, expresses two copies of human SMN2 as wellas two copies of SMN lacking exon 7 (Smn−/−; hSMN2; SMNA7). Due to theseverity of disease in this model, the median survival of this mouse is13 days, which is an insufficient amount of time to assess efficacy ofpotential therapeutic agents.

Treatment with a high dose of SMN-C1, 3 mg/kg/day, results in a mildform of SMA, with the mice appearing largely healthy and displaying onlymodest deficits in body weight and motor function. Specifically,treatment with this high dose of SMN-C1 results in significantcorrection of disease and mimics mild presentations of SMA, such asambulatory Type 111 or Type IV SMA. In this study, at PND24 mice begantreatment with Ab1 (20 mg/kg/week).

Bone Effects

Cortical bone mean crossectional tissue area, mean total crossectionalbone area, mean total crossectional tissue perimeter, mean totalcrossectional bone perimeter, cortical thickness, bone volume, andcortical porosity were measured by micro CT. Trabecular bone volume,trabecular thickness, trabecular number, and trabecularseparation/spacing were also measured.

MicroCT analysis of cortical bone and trabecular bone showedstatistically significant increases in the antibody-treated animals ascompared to control mice. Results are shown in FIGS. 29-31. FIG. 29shows that antibody-treated animals exhibited a statisticallysignificant increase in mean total crossectional bone area and corticalthickness as compared to control (PBS). FIG. 30 shows thatantibody-treated animals exhibited an increase in trabecular bonevolume, trabecular thickness, and trabecular number as compared tocontrol. Additionally, antibody-treated animals showed a decrease intrabecular separation as compared to control.

It is well known in the art that weight-bearing activity is an importantstimulus for bone mass accrual. Surprisingly, animals treated with themyostatin antibody demonstrated not only an increase in weight-bearingbone (FIGS. 29 and 30) but also demonstrated an increase in bone volumein non-weight bearing bone, e.g., the vertebrae (FIG. 31). This increasein non-weight bearing bone further demonstrates that the myostatininhibitors disclosed herein act not only to increase bone volumethrough, for example, increased muscle stimulation, but also act as akey regulator to increase general metabolic effects, including bonehealth.

Muscle Effects

The same pharmacological model of SMA was also used to assess musclefunction in animals treated with an inhibitor of myostatin activationversus control. Total body weight, specific muscle weights, muscleforce, and muscle fiber type and cross sectional areas were measured.FIG. 32 demonstrates that mice treated with Ab1 exhibited a 14.4%increase in body weight at day 50 as compared to control mice (PBStreatment), and FIG. 33 depicts the increase in weight of severalmuscles: gastrocnemius, TA, EDL, soleus, and masseter, after treatmentwith Ab1. FIG. 34A depicts an increase of 23% in plantarflexor force(maximum torque) after treatment with Ab1 versus PBS control, and a 20%increase in plantarflexor force maximum torque/limb length aftertreatment with Ab1 versus PBS control. FIG. 34B depicts masseter forcein Ab1-treated mice as compared to controls.

The differential efficacy of antibody treatment on the gastrocnemius(which constitutes the bulk of the plantarflexor group) and the massetermay be attributed to many facets of SMA disease pathology. For example,among other factors, it is known that myostatin inhibitionpreferentially results in hypertrophy of fast glycolytic muscle fibers(Type IIB in the mouse) (sec PCT/US2017/037332, the entire contents ofwhich are incorporated herein by reference). While the mousegastrocnemius muscle consists primarily of Type IIB fibers (˜75%), themasseter has significantly fewer, between 10 and 25%. Therefore, it isnot surprising that masseter weight and force did not increase aftertreatment with the myostatin antibody.

FIG. 35 depicts histology data from the high-dose SMN-C1 cohorts.Specifically, FIG. 35 shows the total fiber cross sectional area (CSA)and a histogram of CSA distribution in control (vehicle) versus Ab1treated animals, demonstrating an increasing trend in fiber CSA. Thisincrease was attributed entirely to type IIb fibers (data not shown).

While several embodiments of the present disclosure have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the presentdisclosure. More generally, those skilled in the art will readilyappreciate that all parameters, dimensions, materials, andconfigurations described herein are meant to be exemplary and that theactual parameters, dimensions, materials, and/or configurations willdepend upon the specific application or applications for which theteachings of the present disclosure is/are used. Those skilled in theart will recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of thedisclosure described herein. It is, therefore, to be understood that theforegoing embodiments are presented by way of example only and that,within the scope of the appended claims and equivalents thereto, thedisclosure may be practiced otherwise than as specifically described andclaimed. The present disclosure is directed to each individual feature,system, article, material, and/or method described herein. In addition,any combination of two or more such features, systems, articles,materials, and/or methods, if such features, systems, articles,materials, and/or methods are not mutually inconsistent, is includedwithin the scope of the present disclosure.

1. A composition comprising an antibody, or antigen-binding fragmentthereof, that specifically binds pro/latent-myostatin and blocks releaseof mature myostatin, for use as a medicament in treatment or preventionof a metabolic disease in a human subject, comprising steps of:selecting a human subject suffering from or at risk of developing ametabolic disease; and, administering to the human subject thecomposition comprising an effective amount of the antibody, orantigen-binding fragment thereof.
 2. The composition of claim 1, whereinthe subject does not have a myopathy, optionally wherein the myopathy isa primary myopathy or a secondary myopathy.
 3. The composition of claim1, wherein the subject is an adult human subject suffering from growthhormone (GH) deficiency, optionally wherein the subject concurrentlyreceives a recombinant GH therapy or a GH gene therapy.
 4. Thecomposition of claim 1, wherein the metabolic disease is selected fromthe group consisting of type I diabetes, type II diabetes, obesity,metabolic syndrome/pre-diabetes, cardiovascular disease, non-alcoholicsteatohepatitis (NASH), spinal cord injury (SCI), SMA, a hypo-metabolicstate, double diabetes, metabolic bone disorders, Cushings disease, andan obesity syndrome.
 5. The composition of claim 4, wherein thecardiovascular disease is heart failure.
 6. The composition of claim 5,wherein the heart failure is CHF that includes fluid overload.
 7. Thecomposition of claim 6, wherein the fluid overload includes systemicedema and/or pulmonary edema.
 8. The composition of claim 6, wherein thehuman subject responds poorly to diuretic therapy.
 9. The composition ofclaim 4, wherein the obesity is sarcopenic obesity.
 10. The compositionof claim 4, wherein the human subject is on a diet.
 11. The compositionof claim 10, wherein the diet is a caloric restriction diet.
 12. Thecomposition of claim 11, wherein the subject is also physicalactivity-limited.
 13. The composition of any one of claims 10-12,wherein the subject is not on an exercise regimen.
 14. The compositionof any one of claims 10-12, wherein the subject is on an exerciseregimen.
 15. The composition of claim 4, wherein the hypo-metabolicstate is selected from the group consisting of a state associated withprolonged immobilization, a state associated with bed-rest, a stateassociated with casting, a state associated with a stroke, a stateassociated with amputation, and a post-surgery state.
 16. Thecomposition of claim 4, wherein the Cushings disease is selected fromthe group consisting of corticosteroid-induced Cushings disease andtumor-induced Cushings disease.
 17. The composition of claim 4, whereinthe obesity syndromc is selected from the group consisting of PraderWilli, an obesity syndrome associated with a genetic disorder, and anobesity syndrome associated with a hypothalamic disorder.
 18. Thecomposition of any one of claims 1-17, wherein administration of thecomposition causes at least one of the following: a) increases massand/or function of a muscle tissue in the human subject; b) increasesmass and/or function of a fast twitch muscle tissue in the humansubject; c) increases mass and/or function of a slow twitch muscletissue in the human subject; d) increases the metabolic rate of thehuman subject; e) increases insulin sensitivity in the human subject; f)increases the level of brown adipose tissue in the human subject; g)increases the level of beige adipose tissue in the human subject; h)decreases the level of white adipose tissue in the human subject; i)decreases the level of visceral adipose tissue in the human subject; j)decreases the ratio of adipose-to-muscle tissue in the human subject; k)increases glucose uptake by a target tissue in the human subject,wherein the target tissue is selected from the group consisting of brownadipose tissue, beige adipose tissue, and muscle tissue; l) decreasesglucose uptake by a target tissue in the human subject, wherein thetarget tissue is selected from the group consisting of a white adiposetissue and a liver tissue; m) decreases muscle catabolism of proteinand/or muscle release of amino acids in the human subject; n) increasesinsulin dependent glycemic control in the human subject; o) decreasesintramuscular fat infiltration in the human subject; p) improves astandardized quality of life test score; q) prevents muscle loss oratrophy in the human subject; r) increases bone density or volume; s)prevents or reduces bone loss or fracture; t) reduces fluid overload oredema associated with chronic heart failure (CHF); and/or u) enhancesthe subject's responsiveness to a therapy.
 19. The composition of anyone of claims 1-18, wherein the antibody, or antigen-binding portionthereof does not bind to mature myostatin, GDF11 or Activin.
 20. Thecomposition of any one of the foregoing claims, wherein the antibody, orantigen binding fragment thereof, comprises a) a heavy chain variableregion comprising an amino acid sequence of SEQ ID NO:25 and a lightchain variable region comprising an amino acid sequence of SEQ ID NO:31;or b) a heavy chain comprising an amino acid sequence of SEQ ID NO:50and a light chain comprising an amino acid sequence of SEQ ID NO:51.